Wheat with reduced lipoxygenase activity

ABSTRACT

A series of independent human-induced non-transgenic mutations found at one or more of the Lpx genes of wheat; wheat plants having these mutations in one or more of their Lpx genes; and a method of creating and finding similar and/or additional mutations of Lpx by screening pooled and/or individual wheat plants. The wheat plants disclosed herein exhibit decreased lipoxygenase activity without having the inclusion of foreign nucleic acids in their genomes. Additionally, products produced from the wheat plants disclosed herein display increased oxidative stability and increased shelf life without having the inclusion of foreign nucleic acids in their genomes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/182,299 filed Jun. 19, 2015, which is incorporated herein byreference in its entirety.

FIELD

In one embodiment, the disclosure relates to mutations in one or morelipoxygenase 1 (Lpx1) genes. In one embodiment, the disclosure relatesto human-induced non-transgenic mutations in one or more Lpx1 genes ofwheat and wheat plants. In still another embodiment, human-inducednon-transgenic mutations are in the Lpx1 genes in the B or D genome.

In one embodiment, the disclosure relates to wheat plants having wheatseeds and wheat flour with increased oxidative stability as a result ofnon-transgenic mutations in at least one of the Lpx1 genes. In anotherembodiment, the disclosure relates to a method that utilizesnon-transgenic means to create wheat plants having mutations in at leastone of their Lpx1 genes. In yet another embodiment, the disclosurerelates to wheat flour and wheat-based food and beverage products madefrom the seeds of these wheat plants having mutations in at least one oftheir Lpx1 genes.

SUBMISSION OF SEQUENCE LISTING

-   -   The contents of the electronic submission of the text file        Sequence Listing, which is named ARC-38806.txt, which was        created on Sep. 6, 2016, and is 38 KB in size, is incorporated        herein by reference in its entirety.

BACKGROUND

Wheat is an important and strategic cereal crop for the majority of theworld's populations. It is the most important staple food of about twobillion people (36% of the world population). Worldwide, wheat providesnearly 55% of the carbohydrates and 20% of the food calories consumedglobally. It exceeds in acreage and production every other grain crop(including rice, maize, etc.) and is therefore, the most importantcereal grain crop of the world, which is cultivated over a wide range ofclimatic conditions. The understanding of genetics and genomeorganization using molecular markers is of great value for genetic andplant breeding purposes.

The world's main wheat producing regions are China, India, UnitedStates, Russian Federation, France, Australia, Germany, Ukraine, Canada,Turkey, Pakistan, Argentina, Kazakhstan and United Kingdom. Most of thecurrently cultivated wheat varieties belong Triticum aestivum L., whichis known as common bread wheat and valued for bread making. The greatestportion of the wheat flour produced is used for bread making.

Bread wheat is a hexaploid, with three complete genomes termed A, B andD in the nucleus of each cell. Each of these genomes is almost twice thesize of the human genome and consists of around 5,500 millionnucleotides. Durum wheat, also known as macaroni wheat or pasta wheat(Triticum durum or Triticum turgidum subsp. durum), is the majortetraploid species of wheat of commercial importance, which is widelycultivated today. Durum wheat has two complete genomes, A and B, and iswidely used for making pasta.

Wheat is a widely studied plant, but in some cases, development of newtraits is hampered by limited genetic diversity in today's commercialwheat cultivars and also because the bread wheat genome typically hasthree functionally redundant copies of each gene (called homoeologs),and therefore, single gene alterations usually do not produce anyreadily visible phenotype such as those that have been found in diploidcorn. Often in bread wheat, altered variants of all three homoeologsmust be combined genetically in order to evaluate their effects.

Improving the shelf life of whole grain flour is important to meet theincreasing food demands of the world population. Whole grain productsoffer many health advantages such as reducing the risk of chronicdiseases such as coronary heart disease, type 2 diabetes, and some typesof cancer. Whole grain products can also help improve body weightmanagement and digestive health. Despite these health benefits, greaterthan 95% of the United States population consume below the recommendeddaily allowance of whole grains. Consumption of whole grain products arelower due to the bitter and off flavors that develop more rapidly inwhole grain flour due to the susceptibility of its lipid fraction tohydrolytic and oxidative rancidity by lipases, lipoxygenases and otherenzymes. Improving shelf-life and sensory characteristics of whole grainflour and food products by improving oxidative stability couldpositively affect consumer acceptance of whole grain products.

Lipoxygenase (Lpx), linoleate: oxygen oxidoreductase; (EC 1.13.11.12) isa class of non-heme iron-containing dioxygenases that catalyse thepositional and specific dioxygenation of polyunsaturated fatty acidsthat contain 1,4-cis,cis pentadiene structures to produce thecorresponding hydroperoxides. Lpx are key enzymes catalyzing theoxidation of polyunsaturated fatty acids. Lpxs are non-hemeiron-containing dioxygenases, and are monomeric proteins with molecularmass ranging from 94 to 105 kDa in plants. There are many Lpx genes inplant genomes. For example, the Arabidopsis genome contains 6 Lpx genesand the rice genome contains 14 Lpx genes. Wheat, which has a genomesize 108 times larger than Arabidopsis and 36 times larger than rice,has not yet been fully characterized for Lpx genes. At least 3 knownwheat Lpx gene families, each with at least one homoeolog on the A, Band D genomes, have been identified to date. Lpx1 and Lpx3 are onchromosome 4 and Lpx2 is on chromosome 5. A quantitative trait locus forlipoxygenase activity has also recently been reported on chromosome 1Ain durum wheat.

In plants, products of the lipoxygenase reaction have been shown to haveroles in several processes, such as vegetative growth, wounding,response to herbivore and pathogen attack and also mobilization ofstorage lipids during germination. In rice, double mutants of twodifferent genes, Lox1 and Lox2, but not single mutations in Lox3,improved germination and stability of intact grains for up to 42 months.

In durum wheat, radicals produced during the intermediate states ofpolyunsaturated fatty acid hydroperoxidation can cause oxidation ofcarotenoid pigments, and consequently a loss of the yellow flour colorpreferred for pasta products. Wheat lipoxygenases have beencharacterized in durum wheat due to efforts to increase yellowcarotenoid levels in those varieties. A deletion allele in the durumwheat LpxB1.1 gene (called Lpx-B1.1c) in particular was found associatedwith improved yellow color in pasta products. Durum wheat lines with lowlipoxygenase activity were also associated with a reduction of Lpx-3transcript levels in the late stages of grain filling.

In addition to the LpxB1.1 gene, the wheat B genome has an additionalcopy of the Lpx1 gene that is present either as LpxB1.2 or LpxB1.3. Inboth durum and bread wheat, the A genome Lpx1 gene is encoded by apseudogene called LpxA1-like (GenBank FJ518909). Durum wheat does nothave the D genome, but an Lpx1 gene in the D genome of bread wheat hasalso been identified, and the sequence recently deposited in GenBank(KC679302).

In bread wheat, lipases and lipoxygenases play a role in lipiddegradation, which can contribute to wheat products with decreasednutritional quality, decreased functional properties and decreasedsensory acceptability. Lipoxygenase activity in bread wheat leads todegradation of carotenoids and decreased nutritional value. Sincemultiple Lpx genes (Lpx1, 2 and 3) are all expressed in the wheat graineach with one or more potential homoeologs in the A, B and D genomes, itis unclear if altering one gene or gene family could positively affectoxidative stability of whole grain flour in bread wheat. Mutations inthe lipoxygenase genes in the wheat genome provide a potential pathwayfor providing increased oxidative stability in wheat flour and productsderived therefrom. The disclosure herein demonstrates that novel allelesin the Lpx1 gene significantly improve shelf-life of whole grain flour.

SUMMARY

In one embodiment, the disclosure relates to mutations in one or morelipoxygenase 1 (Lpx1) genes. In one embodiment, the disclosure relatesto non-transgenic mutations in one or more Lpx1 genes. In oneembodiment, one or more mutations are in the Lpx1 gene of the B genome.In another embodiment, one or more mutations are the Lpx1 gene of the Dgenome.

In one embodiment, the disclosure relates to multiple non-transgenicmutations in the Lpx gene including but not limited to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, and greater than 10 mutations.

In one embodiment, the disclosure relates to non-transgenic mutations inthe Lpx-B1.2 gene of the B genome including but not limited to 1, 2, 3,4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In one embodiment, the disclosure relates to non-transgenic mutations inthe Lpx-D1 gene of the D genome including but not limited to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In another embodiment, the disclosure relates to non-transgenicmutations in the Lpx B1.2 gene of the B genome including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and non-transgenic mutations in theLpx-D1 gene of the D genome including but not limited to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, and greater than 10 mutations.

In another embodiment, the disclosure relates to a wheat plant, wheatseeds, wheat plant parts, and progeny thereof with decreasedlipoxygenase activity compared to wild type wheat plant, wheat seeds,wheat plant parts, and progeny thereof.

In another embodiment, the disclosure relates to a wheat plant, wheatseeds, wheat plant parts, and progeny thereof with increased oxidativestability compared to wild type wheat plant, wheat seeds, wheat plantparts, and progeny thereof.

In another embodiment, this invention relates to a wheat plant, wheatseeds, wheat plant parts, and progeny thereof having reducedlipoxygenase activity compared to the wild type wheat plant, wherein thereduction in lipoxygenase activity is caused by a human-inducednon-transgenic mutation in one or more of the wheat plant's Lpx1 genes.In another embodiment, the Lpx1 enzyme has reduced activity.

In another embodiment, the disclosure relates to a wheat plantcontaining one or more mutated Lpx1 genes, as well as seeds, pollen,plant parts and progeny of that plant.

In another embodiment, the disclosure relates to wheat seeds and wheatflour with increased shelf life and improved sensory characteristicshaving reduced Lpx1 enzyme activity caused by a human-inducednon-transgenic mutation in one or more Lpx1 genes.

In another embodiment, the disclosure relates to food, beverage, andfood and beverage products incorporating wheat seeds and wheat flourhaving reduced Lpx1 enzyme activity caused by a human-inducednon-transgenic mutation in one or more Lpx1 genes.

In another embodiment, this disclosure relates to a wheat plant havingreduced activity of one or more Lpx1 enzymes compared to the wild typewheat plants, created by the steps of obtaining plant material from aparent wheat plant, inducing at least one mutation in at least one copyof an Lpx1 gene of the plant material by treating the plant materialwith a mutagen to create mutagenized plant material (e.g., seeds orpollen), analyzing progeny wheat plants to detect at least one mutationin at least one copy of an Lpx1 gene, selecting progeny wheat plantsthat have at least one mutation in at least one copy of an Lpx1 gene,and optionally, crossing progeny wheat plants that have at least onemutation in at least one copy of an Lpx1 gene with other progeny wheatplants that have at least one mutation in a different copy of an Lpx1gene, and repeating the cycle of identifying progeny wheat plants havingmutations and optionally crossing the progeny wheat plants havingmutations with other progeny wheat plants having mutations to produceprogeny wheat plants with reduced Lpx1 enzyme activity. In anotherembodiment, the method comprises growing or using the mutagenized plantmaterial to produce progeny wheat plants.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases. Onlyone strand of each nucleic acid sequence is shown, but the complementarystrand is understood to be included by any reference to the displayedstrand. In the accompanying sequence listing:

SEQ ID NO: 1 shows a Triticum aestivum gene for Lipoxygenase 1, Dgenome, Lpx-D1 exons 1-6 (3,863 base pairs).

SEQ ID NO: 2 shows the Lpx-D1 coding sequence of SEQ ID NO: 1 (2,586base pairs).

SEQ ID NO: 3 shows the Lpx-D1 protein sequence of SEQ ID NO. 2 (862amino acids).

SEQ ID NO: 4 shows a Triticum aestivum gene for Lipoxygenase 1, Bgenome, Lpx-B1.2 exons 1-7 (4,263 base pairs).

SEQ ID NO: 5 shows the Lpx-B1.2 coding sequence of SEQ ID NO. 4 (2,586base pairs).

SEQ ID NO: 6 shows the Lpx-B1.2 protein sequence of SEQ ID NO. 5 (862amino acids).

SEQ ID NOs: 7-14 show exemplary homoeolog specific primers that haveproven useful in identifying useful mutations within the Lpx-D1 andLpx-B1.2 gene sequences.

SEQ ID NO: 15 shows the Lpx-D1 promoter sequence and first exon for SEQID NO:1 SEQ ID NO: 16-17 show exemplary homoeolog specific primers thathave proven useful in identifying useful mutations within the Lpx-D1promoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph showing the increase in hexanal production ofwhole grain flour as an indicator of rancidity during an acceleratedaging time course at 37° C.

FIG. 2 is a bar graph showing the improved shelf life of novel Lpx-D1and Lpx-B1.2×Lpx-D1 alleles during an accelerated aging time course at37° C.

FIG. 3 is a bar graph showing reduced rancidity and improved shelf-lifein whole grain flour of multiple Lpx-D1 alleles during an 8 weeksaccelerated aging time-course at 37° C.

FIG. 4 is a bar graph showing reduced rancidity and improved shelf-lifeof novel Lpx-D1 and Lpx-B1.2/Lpx-D1 alleles during an 8 weeksaccelerated aging time course at 37° C. Triplicate biological replicateswere assayed and analyzed for hexanal levels.

FIG. 5 is a bar graph showing the improved shelf-life of novel Lpx-D1and Lpx-B1.2×Lpx-D1 alleles during an accelerated aging time-course at37° C. for 12 and 30 weeks (equivalent to 10 and 24 months at roomtemperature, respectively).

FIG. 6 is a bar graph showing reduced bitter compound, pinellic acid, inaged whole grain flour made from grains with Lpx-D1 novel alleles.

FIG. 7 is a bar graph showing reduced levels of bitter compound,pinellic acid, in dough made from freshly milled or aged whole grainflour from grains with Lpx1 novel alleles.

DETAILED DESCRIPTION Definitions

The numerical ranges in this disclosure are approximate, and thus mayinclude values outside of the range unless otherwise indicated.Numerical ranges include all values from and including the lower and theupper values, in increments of one unit, provided that there is aseparation of at least two units between any lower value and any highervalue. As an example, if a compositional, physical or other property,such as, for example, molecular weight, viscosity, etc., is from 100 to1,000, it is intended that all individual values, such as 100, 101, 102,etc., and sub ranges, such as 100 to 144, 155 to 170, 197 to 200, etc.,are expressly enumerated. For ranges containing values which are lessthan one or containing fractional numbers greater than one (e.g., 1.1,1.5, etc.), one unit is considered to be 0.0001, 0.001, 0.01 or 0.1, asappropriate. For ranges containing single digit numbers less than ten(e.g., 1 to 5), one unit is typically considered to be 0.1. These areonly examples of what is specifically intended, and all possiblecombinations of numerical values between the lowest value and thehighest value enumerated, are to be considered to be expressly stated inthis disclosure. Numerical ranges are provided within this disclosurefor, among other things, relative amounts of components in a mixture,and various temperature and other parameter ranges recited in themethods.

As used herein, the term “allele” is any of one or more alternativeforms of a gene, all of which relate to one trait or characteristic. Ina diploid cell or organism, the two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes. An allele can be“wild-type” indicating the parental sequence at a particular nucleotideposition, or “mutant” indicating a different nucleotide than theparental sequence. The term “heterozygous” indicates one wild-type andone mutant allele at a particular nucleotide position, and the term“homozygous” indicates two of the same allele at a particular nucleotideposition.

As used herein, the terms “altering”, “increasing”, “increased”,“reducing”, “reduced”, “inhibited” or the like are considered relativeterms, i.e. in comparison with the wild-type or unaltered state. The“level of a protein” refers to the amount of a particular protein, forexample Lpx, which may be measured by any means known in the art suchas, for example, Western blot analysis, or mass spectrometry or otherimmunological means. The “level of an enzyme activity” refers to theamount of a particular enzyme measured in an enzyme assay. It would beappreciated that the level of activity of an enzyme might be altered ina mutant but not the expression level (amount) of the protein itself.Conversely, the amount of protein might be altered but the activityremain the same if a more or less active protein is produced. Reductionsin both amount and activity are also possible such as, for example, whena gene encoding the enzyme is inactivated. In certain embodiments, thereduction in the level of protein or activity is by at least 10% or byat least 20% or by at least 30% or by at least 40% or by at least 50% orby at least 60% compared to the level of protein or activity in theendosperm of unmodified wheat, or by at least 70%, or by at least 80% orby at least 85% or by at least 90% or at least 95%. The reduction in thelevel of the protein or enzyme activity or gene expression may occur atany stage in the development of the grain, particularly during the grainfilling stage, or at all stages of grain development through tomaturity.

As used herein, amino acid or nucleotide sequence “identity” and“similarity” are determined from an optimal global alignment between thetwo sequences being compared. An optimal global alignment is achievedusing, for example, the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48:443-453). Sequences may also be alignedusing algorithms known in the art including but not limited to CLUSTAL Valgorithm or the BLASTN or BLAST 2 sequence programs.

“Identity” means that an amino acid or nucleotide at a particularposition in a first polypeptide or polynucleotide is identical to acorresponding amino acid or nucleotide in a second polypeptide orpolynucleotide that is in an optimal global alignment with the firstpolypeptide or polynucleotide. In contrast to identity, “similarity”encompasses amino acids that are conservative substitutions. A“conservative” substitution is any substitution that has a positivescore in the Blosum62 substitution matrix (Henikoff and Henikoff, 1992,Proc. Natl. Acad. Sci. USA 89: 10915-10919).

By the statement “sequence A is n % similar to sequence B,” it is meantthat n % of the positions of an optimal global alignment betweensequences A and B consists of identical residues or nucleotides andconservative substitutions. By the statement “sequence A is n %identical to sequence B,” it is meant that n % of the positions of anoptimal global alignment between sequences A and B consists of identicalresidues or nucleotides.

As used herein, “increase in shelf life” refers to an increase in thetime period for which the product can remain sellable or useable. Forexample, millers commonly stamp ‘use by’ dates after milling for wholegrain flour in the United States. A typical “use by” date may be fourmonths. An “increase in shelf life” would extend the use by date.

As used herein, the term “plant” includes reference to an immature ormature whole plant, including a plant from which seed or grain oranthers have been removed. A seed or embryo that will produce the plantis also considered to be the plant.

As used herein, the term “plant parts” includes plant protoplasts, plantcell tissue cultures from which wheat plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants, such as embryos, pollen, ovules, pericarp, seed, flowers,florets, heads, spikes, leaves, roots, root tips, anthers, and the like.

As used herein, the term “polypeptide(s)” refers to any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds. “Polypeptide(s)” refers to bothshort chains, commonly referred to as peptides, oligopeptides andoligomers, and to longer chains generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptide(s)” include those modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature and they arewell known to those of skill in the art. It will be appreciated that thesame type of modification may be present in the same or varying degreeat several sites in a given polypeptide.

As used herein, an “Lpx1 derivative” refers to a Lpx1protein/peptide/polypeptide sequence that possesses biological activitythat is substantially reduced as compared to the biological activity ofthe whole Lpx1 protein/peptide/polypeptide sequence. In other words, itrefers to a polypeptide of a modified Lpx1 protein that has reduced Lpx1enzymatic activity. The term “Lpx1 derivative” encompasses the“fragments” or “chemical derivatives” of a modified Lpx1protein/peptide.

As used herein, the term “polynucleotide(s)” generally refers to anypolyribonucleotide or poly-deoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. This definition includes, withoutlimitation, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions or single-, double- andtriple-stranded regions, cDNA, single- and double-stranded RNA, and RNAthat is a mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded, or triple-stranded regions, or a mixture ofsingle- and double-stranded regions. The term “polynucleotide(s)” alsoembraces short nucleotides or fragments, often referred to as“oligonucleotides,” that due to mutagenesis are not 100% identical butnevertheless code for the same amino acid sequence.

A “reduced or non-functional fragment,” as is used herein, refers to anucleic acid sequence that encodes for a Lpx1 protein that has reducedbiological activity as compared the protein coding of the whole nucleicacid sequence. In other words, it refers to a nucleic acid orfragment(s) thereof that substantially retains the capacity of encodinga Lpx1 polypeptide of the invention, but the encoded Lpx1 polypeptidehas reduced activity.

The term “fragment,” as used herein, refers to a polynucleotidesequence, (e.g, a PCR fragment) which is an isolated portion of thesubject nucleic acid constructed artificially (e.g., by chemicalsynthesis) or by cleaving a natural product into multiple pieces, usingrestriction endonucleases or mechanical shearing, or a portion of anucleic acid synthesized by PCR, DNA polymerase or any otherpolymerizing technique well known in the art, or expressed in a hostcell by recombinant nucleic acid technology well known to one of skillin the art.

With reference to polynucleotides of the disclosure, the term “isolatedpolynucleotide” is sometimes used. This term, when applied to DNA,refers to a DNA molecule that is separated from sequences with which itis immediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it was derived. For example,the “isolated polynucleotide” may comprise a PCR fragment. In anotherembodiment, the “isolated polynucleotide” may comprise a DNA moleculeinserted into a vector, such as a plasmid or virus vector, or integratedinto the genomic DNA of a prokaryote or eukaryote. An “isolatedpolynucleotide molecule” may also comprise a cDNA molecule.

A wheat plant is defined herein as any plant of a species of the genusTriticum, which species is commercially cultivated, including, forexample, Triticum aestivum L. ssp. aestivum (common or bread wheat),other subspecies of Triticum aestivum, Triticum turgidum L. ssp. durum(durum wheat, also known as macaroni or hard wheat), Triticum monococcumL. ssp. monococcum (cultivated einkorn or small spelt), Triticumtimopheevi ssp. timopheevi, Triticum turgigum L. ssp. dicoccon(cultivated emmer), and other subspecies of Triticum turgidum (Feldman).The wheat may be hexaploid wheat having an AABBDD type genome, ortetraploid wheat having an AABB type genome. Since genetic variation inwheat transferred to certain related species, including rye and barleyby hybridization, the disclosure also includes the hybrid species thusformed, including triticale that is a hybrid between bread wheat andrye. In one embodiment, the wheat plant is of the species Triticumaestivum, and preferably of the subspecies aestivum. Alternatively,since mutations or transgenes can be readily transferred from Triticumaestivum to durum wheat, the wheat is preferably Triticum turgidum L.ssp. Durum.

In one embodiment, the disclosure relates to non-transgenic mutations inone or more Lpx1 genes. In another embodiment, the disclosure describeswheat plants exhibiting seeds with deceased Lpx1 activity as compared towild type wheat seeds without the inclusion of foreign nucleic acids inthe wheat plant genome. In yet another embodiment, the disclosuredescribes wheat plants exhibiting seeds with increased oxidativestability as compared to wild type wheat seeds, without the inclusion offoreign nucleic acids in the wheat plant genome. In yet anotherembodiment, the disclosure describes wheat plants exhibiting seedsproducing flour with increased shelf life as compared to wild type wheatseeds, without the inclusion of foreign nucleic acids in the wheat plantgenome.

In still another embodiment, the disclosure relates to a series ofindependent human-induced non-transgenic mutations in one or more Lpx1genes; wheat plants having one or more of these mutations in at leastone Lpx1 gene thereof; and a method of creating and identifying similarand/or additional mutations in at least one Lpx1 gene of wheat.Additionally, the disclosure relates to wheat plants exhibiting seedwith decreased Lpx1 activity and/or increased oxidative stability and/orshelf life as compared to wild type wheat seed, without the inclusion offoreign nucleic acids in the plants' genomes.

I. Lpx1 Mutations

A. Lpx1 Genes

In one embodiment, the disclosure relates to one or more non-transgenicmutations in the Lpx1 genes including the promoter. In anotherembodiment, the invention relates to one or more mutations in the Lpx1gene. In one embodiment, the disclosure relates to multiplenon-transgenic mutations in the Lpx1 gene including but not limited to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In another embodiment, the Lpx1 gene may contain one or morenon-transgenic mutations recited in Tables 1, 2 and 3 and correspondingmutations in homoeologues and combinations thereof.

In another embodiment, the disclosure relates to corresponding mutationsto the one or more non-transgenic mutations disclosed herein in the Lpx1gene in a corresponding homoeologue. By way of example, an identifiedmutation in the Lpx-D1 gene of the D genome may be a beneficial mutationin the Lpx1 gene of the B and/or A genome. One of ordinary skill in theart will understand that the mutation in the homoeologue may not be inthe exact location.

One of ordinary skill in the art understands there is natural variationin the genetic sequences of the Lpx1 genes in different wheat varieties.

The inventors have determined that to achieve a oxidative stability inwheat plants, mutations that reduce Lpx1 gene function are desirable.Preferred mutations include missense and nonsense changes, includingmutations that prematurely truncate the translation of one or more Lpx1proteins from messenger RNA, such as those mutations that create a stopcodon within the coding region of an Lpx1 messenger RNA. Such mutationsinclude insertions, deletions, repeat sequences, splice junctionmutations, modified open reading frames (ORFs) and point mutations. Somestop codon mutations are more effective than others because not all stopcodon mutations reduce lipoxygenase activity to the same extent.

In still another embodiment, one or more mutations are in the Lpx-B1.2gene of the B genome. In still another embodiment, one or more mutationsare in the Lpx-D1 gene of the D genome. In another embodiment, one ormore mutations are in the Lpx-B1.2 and Lpx-D1 genes of the B and Dgenomes.

1. B Genome

In one embodiment, the disclosure relates to multiple non-transgenicmutations in the Lpx gene of the B genome including but not limited to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations. In oneembodiment, one or more non-transgenic mutations are in both alleles ofthe Lpx gene in the B genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the Lpx gene of the B genome.

In one embodiment, one or more mutations are in the Lpx-B1.1a gene ofthe B genome. In one embodiment, the disclosure relates to multiplenon-transgenic mutations in the Lpx-B1.1a gene including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In one embodiment, one or more mutations are in the Lpx-B1.1b gene ofthe B genome. In one embodiment, the disclosure relates to multiplenon-transgenic mutations in the Lpx-B1.1b gene including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In one embodiment, one or more mutations are in the Lpx-B1.1c gene ofthe B genome. In one embodiment, the disclosure relates to multiplenon-transgenic mutations in the Lpx-B1.1c gene including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In one embodiment, one or more mutations are in the Lpx-B1.2 gene of theB genome. In one embodiment, the disclosure relates to multiplenon-transgenic mutations in the Lpx-B1.2 gene including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In one embodiment, one or more mutations are in the Lpx-B1.3 gene of theB genome. In one embodiment, the disclosure relates to multiplenon-transgenic mutations in the Lpx-B1.3 gene including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

The following mutations identified in Tables 1, 2 and 3 are exemplary ofthe mutations created and identified according to various embodimentsdisclosed herein. They are offered by way of illustration, notlimitation. It is to be understood that the mutations below are merelyexemplary and that similar mutations are also contemplated.

One exemplary mutation in Table 1 is G2982A, resulting in a change fromguanine to adenine at nucleotide position 2982 identified according toits position in the sequence of Lpx-B1.2 SEQ ID NO: 4. This mutationresults in a change from tryptophan to a stop (*) codon at amino acidposition 510 (W510*) identified according to its position in theexpressed protein of Lpx-B1.2 (SEQ ID NO: 6).

Table 1 provides examples of mutations created and identified inLpx-B1.2 in the B genome of wheat plants, variety Express. Nucleotideand amino acid changes are identified according to their positions inSEQ ID NOs: 4 and 6, respectively. Zygosity refers to whether themutation is heterozygous (Het) or homozygous (Hom) in the M2 plant.

TABLE 1 Representative mutations in the Lpx-B1.2 gene in the B genomeNucleotide A.A. Change Mutation Primer (SEQ ID (SEQ ID SEQ IDs NO: 4)NO: 6) PSSM SIFT Description Zygosity 8, 14 G2536A V397I −12.9 1.001705D03 Hom 8, 14 C2563T L406F 2316C07 Hom 8, 14 G2691A Splice Junction2171F09 Hom 8, 14 C2695T L415F 2330B06 Hom 8, 14 G2722A D424N 1705E09Hom 8, 14 C2728T H426Y 2194G09 Het 8, 14 C2749T L433F 2333E09 Het 8, 14C2770T P440S 1730E05 Het 8, 14 C2771T P440L 2177E04 Het 8, 14 C2800TL450F 8.1 0.06 2193D10 Hom 8, 14 T2810A L453Q 22.4 0.00 1711F03 Het 8,14 G2816A G455D −4.6 1.00 2178B03 Hom 8, 14 G2818A D456N 5.1 0.262179F05 Het 8, 14 G2822A G457D 14.6 0.02 1705F02 Het 8, 14 G2825A R458K4.4 0.00 2171F04 Hom 8, 14 C2831T T460M −2.1 0.04 2196D10 Het 8, 14C2833T P461S 26.1 0.01 2169G03 Het 8, 14 G2839A A463T 20.8 0.01 2177D04Hom 8, 14 G2854A E468K −3.0 1.00 1743D06 Het 8, 14 C2858T P469L 28.00.00 2179B06 Hom 8, 14 C2879T T476I 2171G11 Het 8, 14 C2882T T477I2328B06 Hom 8, 14 G2884A A478T 2196D11 Het 8, 14 C2885T A478V 2328E04Het 8, 14 C2903T T484M 1744F11 Het 8, 14 G2918A G489D 2322F06 Hom 8, 14G2921A S490N 1706A05 Hom 8, 14 G2926A E492K 2330A10 Het 8, 14 G2929AG493R 2328A08 Het 8, 14 G2933A W494* 1721E07 Hom 8, 14 G2941A E497K −0.50.93 1712C12 Het 8, 14 T2944G F498V 5.5 0.01 1706E05 Hom 8, 14 C2948TA499V 21.1 0.00 2178D09 Hom 8, 14 G2962A A504T 4.1 0.56 2322E05 Het 8,14 C2963T A504V 3.0 0.41 1705C08 Het 8, 14 G2971A D507N 8.4 0.12 2172H04Het 8, 14 C2975T S508F −2.9 0.82 2195C02 Hom 8, 14 G2977A G509R 12.50.33 1705B01 Het 8, 14 G2982A W510* 2175D04 Het

In one embodiment, the disclosure relates to a polynucleotide of theLpx-B1.2 gene in the B genome with one or more non-transgenic mutationslisted in Table 1 and corresponding to SEQ ID NO: 4. In anotherembodiment, the polynucleotide with one or more non-transgenic mutationslisted in Table 1 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 4.In yet another embodiment, the polynucleotide with one or morenon-transgenic mutations listed in Table 1 is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99%similar to SEQ ID NO: 4.

In still another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 1 codes for a Lpx-B1.2 protein,wherein the Lpx-B1.2 protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 6. Instill another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 1 codes for a Lpx-B1.2 protein,wherein the Lpx-B1.2 protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO: 6.

2. D Genome

In one embodiment, the disclosure relates to multiple non-transgenicmutations in the Lpx-D1 gene of the D genome including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations. In oneembodiment, one or more non-transgenic mutations are in both alleles ofthe Lpx-D1 gene in the D genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the Lpx-D1gene of the D genome.

In one embodiment, one or more mutations are in the Lpx-D1 gene of the Dgenome. In one embodiment, the disclosure relates to multiplenon-transgenic mutations in the Lpx-D1 gene including but not limited to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

One exemplary mutation in Table 2 is G2629A, resulting in a change fromguanine to adenine at nucleotide position 2629 identified according toits position in the sequence of Lpx-D1 SEQ ID NO: 1. This mutationresults in a change from tryptophan to a stop (*) codon at amino acidposition 494 (W494*) identified according to its position in theexpressed Lpx-D1 protein (SEQ ID NO: 3).

Table 2 provides representative examples of mutations created andidentified in Lpx-D1 in the D genome of wheat plants, variety Express.Nucleotide and amino acid changes are identified according to theirpositions in SEQ ID NOs: 1 and 3, respectively. Zygosity refers towhether the mutation is heterozygous (Het) or homozygous (Hom) in the M2plant.

TABLE 2 Representative mutations in the Lpx-D1 gene in the D genomeNucleotide A.A. Mutation Mutation Primer (SEQ ID (SEQ ID SEQ IDs NO: 1)NO: 3) PSSM SIFT Description Zygosity 10, 11 G592A G70E 1404C02 Het 10,11 G626A W81* 1686F04 Hom 10, 11 G627A V82M 4543C04 Het 10, 11 C631TT83M 2171A08 Het 10, 11 G685A W101* 1686E09 Hom 10, 11 G693A E104K2316D03 Het 10, 11 C709T P109L 4831G10 Hom 10, 11 G712A G110D 2322A03Hom 10, 11 C742T S120F 4831B07 Het 10, 11 C753T L124F 4818D09 Het 10, 11C780T P133S 1685C06 Hom 10, 11 C781T P133L 4547F12 Het 10, 11 G789AG136S 2171A10 Het 10, 11 C795T L138F 2165E05 Het 10, 11 C799T S139F1745F10 Hom 10, 11 C1516T R185W 1737C05 Het 10, 11 C1451T T163M 16.40.01 2319H03 Het 10, 11 G1588A G209S 12.9 0.05 2176A11 Hom 10, 11 C1471TP170S 26.2 0.00 1333E02 Het 10, 11 C1472T P170L 28.2 0.00 2195F07 Het10, 11 G1498A D179N 4831E12 Het 10, 11 G1646A R228H 22.2 0.00 4544B07Het 10, 11 G1517A R185Q 2169E08 Hom 10, 11 G1519A G186S 2164D07 Hom 10,11 G1520A G186D 2172E07 Hom 10, 11 C1531T Q190* 2172A05 Hom 10, 11G1534A G191R 2196D06 Het 10, 11 C1538T P192L 2167C03 Het 10, 11 G1546AE195K 2168C11 Het 10, 11 G1556A R198H 24.0 0.00 4831D09 Hom 10, 11G1558A V199I −2.4 1.00 1745A09 Het 10, 11 C1832T P290L 11.4 0.25 2162B10Hom 10, 11 C1587T L208= 2165H01 Hom 10, 11 G1591A E210K 7.9 0.15 2322B05Het 10, 11 G1966A G335S 2322B05 Het 10, 11 C1600T P213S 2166H07 Het 10,11 C1601T P213L 1333G02 Hom 10, 11 C1987T P342S 1402C09 Het 10, 11G1609A G216S 1334G03 Hom 10, 11 G1610A G216D 1344F01 Hom 10, 11 C1633TP224S 25.6 0.00 1333D05 Hom 10, 11 G1642A G227S 18.2 0.49 2175A08 Hom10, 11 G1651A G230S 13.2 0.04 2166C06 Het 10, 11 C1661T P233L 14.7 0.062189H08 Hom 10, 11 C1675T P238S 13.0 0.09 1738G07 Hom 10, 11 G1679AS239N −5.7 1.00 1344D02 Het 10, 11 G1684A E241K 12.1 0.05 2163H01 Het10, 11 G1691A R243Q 4544B01 Hom 10, 11 C1720T P253S 2191E11 Hom 10, 11C1721T P253L 4818F12 Hom 10, 11 C1723T R254W 1738B10 Het 10, 11 G1724AR254Q 2171B11 Het 10, 11 G1738A G259S 2324H10 Het 10, 11 C1744T L261F2335C01 Het 10, 11 C1772T S270F 4543H09 Hom 10, 11 G1780A A273T 2176C11Het 10, 11 G2032A D357N 10.5 0.17 2176C11 Het 10, 11 C1805T A281V2169B04 Het 10, 11 C1814T T284I 2320B12 Het 10, 11 G1819A V286I 1737E05Hom 10, 11 C1829T T289I 2322E10 Hom 10, 11 C1831T P290S 4.9 0.83 2191F11Hom 10, 11 G1863A M300I 4.6 0.40 2323A10 Het 10, 11 G1873A E304K 4.50.34 1686D09 Hom 10, 11 G1880A G306D 13.1 0.04 1344E07 Hom 10, 11 G1909AE316K 1738D03 Hom 10, 11 C1930T P323S 2320A07 Hom 10, 11 A2051T E363V10.1 0.50 2175E03 Het 7, 9 C2279T L409F 1703B07 Het 7, 8 G2386A SpliceJunction 2196A02 Het 7, 8 G2397A S417N 1702D04 Het 7, 9 C2414T L423F1355F01 Hom 7, 9 C2423T H426Y 1703D03 Het 7, 8 G2430A R428Q 2168H07 Het7, 8 C2438T P431S 2190C02 Het 7, 8 G2450A E435K 1721F01 Hom 7, 8 G2453AV436I 1722C08 Hom 7, 8 C2466T P440L 2169B06 Hom 7, 9 G2468A G441S1459G11 Hom 7, 8 C2487T T447I 20.6 0.00 1729F08 Het 7, 9 G2490A R448K24.0 0.00 1687D06 Hom 7, 9 G2508A R454H 12.7 0.11 1457C09 Hom 7, 8C2528T P461S 26.1 0.01 1721E07 Het 7, 9 C2529T P461L 9.7 0.26 1456D06Het 12, 13 G2549A E468K −3.0 1.00 2334B03 Hom 7, 9 C2553T P469L 28.00.00 1705F03 Het 7, 9 G2564A G473S 1743G02 Het 12, 13 C3306T L677F 11.40.05 4752E11 Het 12, 13 C2574T T476I 2330D08 Hom 7, 8 C2577T T477I1717E01 Het 7, 9 G2591A V482M 1333B11 Hom 7, 9 C2600T P485S 1686A07 Hom12, 13 C2606T P487S 2317A11 Hom 12, 13 G2610A S488N 2334D05 Het 7, 9G2613A G489D 1339F04 Hom 12, 13 G2616A S490N 4776G10 Het 12, 13 G2624AG493S 1748G05 Het 12, 13 G2625A G493D 4752H09 Het 7, 9 G2629A W494*1416F05 Het 7, 9 G2636A E497K −0.5 0.93 1704D07 Hom 12, 13 C2658T A504V3.0 0.41 2335E07 Hom 7, 9 G2666A D507N 8.4 0.12 1689A03 Het 12, 13T2675A W510R 4.0 0.37 2317A08 Hom 12, 13 G2676A W510* 2197F06 Het 12, 13G2677A W510* 1748B07 Het 7, 9 C2684T L513F 6.1 0.12 1686B10 Hom 12, 13G2827A Splice Junction 2319D08 Hom 12, 13 G2828A W517* 2161C05 Het 12,13 G2841A A522T 4.2 0.24 4543C12 Het 7, 9 G2849A M524I 2.5 0.47 1339E01Het 7, 9 G2850A E525K 18.1 0.01 1464D10 Hom 7, 9 C2853T P526S 13.9 0.081705D11 Het 12, 13 G2875A R533Q 25.7 0.00 4543C06 Hom 7, 9 G2884A S536N17.7 0.15 1703G05 Hom 12, 13 C2895T P540S 25.5 0.00 2316E11 Hom 7, 9G2898A V541M 10.6 0.02 1333A08 Hom 12, 13 C2919T H548Y 25.9 0.00 4546A09Het 12, 13 C2932T T552I 1427B11 Hom 12, 13 G3252A D659N 27.6 0.004548F10 Het 12, 13 A2944T N556I 2189H07 Hom 12, 13 G2946A A557T 1428A10Hom 12, 13 C2953T A559V 4551F08 Hom 12, 13 C2955T R560W 1435F06 Het 7, 9G2956A R560Q 1334G09 Het 12, 13 G2993A M572I 4684G04 Het 7, 9 C3003TP576S 1711C07 Het 12, 13 G3022A G582E 2320E08 Het 12, 13 G3049A W591*4549G07 Het 12, 13 G3060A E595K 10.4 0.36 4546D04 Hom 12, 13 C3072TP599S 17.1 0.03 2161B07 Hom 12, 13 C3073T P599L 11.6 0.13 1748A11 Het12, 13 G3105A E610K 2319H09 Het 12, 13 G3108A D611N 4684E07 Het 12, 13G3133A R619Q 2328H04 Hom 12, 13 C3160T A628V 19.0 0.03 4548E09 Hom 12,13 G3162A A629T 3.6 0.39 4544D10 Hom 12, 13 G3165A D630N 11.7 0.064544C09 Hom 12, 13 G3181A W635* 2189B08 Het 12, 13 G3204A G643S −4.50.92 1444C10 Hom 12, 13 G3207A E644K 8.5 0.36 1402C09 Het 12, 13 G3216AA647T 3.9 0.44 4818F10 Het 12, 13 C3256T T660M 9.0 0.22 2319B05 Hom 12,13 G3272A W665* 4544H06 Hom 12, 13 G3275A W666* 1435D10 Hom 12, 13G3282A A669T 5.2 0.05 2330C04 Hom 12, 13 G3288A E671K 7.7 0.45 1749D11Hom 12, 13 G3295A G673E 22.6 0.00 1402B04 Hom 12, 13 C3297T H674Y 15.00.71 1402C07 Hom 12, 13 C3318T P681S 12.8 0.71 2330B06 Het 12, 13 G3345AG690R 2330B06 Het 12, 13 C3319T P681L 16.2 0.30 2161G09 Hom 12, 13G3339A G688S 4543D12 Het 12, 13 G3364A C696Y 22.1 0.00 2194A08 Hom 12,13 C3370T T698I 1.8 0.34 2319D05 Hom 12, 13 G3379A W701* 4543G08 Hom 12,13 G3385A G703E 5.9 0.01 2189D07 Hom 12, 13 G3390A A705T 14.9 0.022334D08 Hom 12, 13 G3399A A708T 10.9 0.06 4544E04 Het 12, 13 C3400TA708V 19.8 0.00 2330E08 Hom 12, 13 G3415A G713E 18.0 0.01 4547D06 Het12, 13 G3432A G719R 14.4 0.01 2193G12 Hom 12, 13 C3438T L721F −1.9 0.302197A05 Hom 12, 13 C3441T P722S 17.4 0.01 2197D11 Hom 12, 13 C3454TT726M 13.4 0.05 1444B07 Hom 12, 13 G3456A V727M −1.3 0.27 2334A02 Hom12, 13 C3462T R729W 6.1 0.08 2330A11 Het 12, 13 C3481T P735L 4551G10 Het12, 13 G3483A G736R 2193F12 Hom 12, 13 G3484A G736E 2320B08 Hom 12, 13G3492A A739T 1444B06 Het 12, 13 G3498A A741T 1428C06 Hom 12, 13 G3501AE742K 4545B08 Hom 12, 13 G3513A D746N 4551F09 Het 12, 13 C3517T P747L4549D10 Het 12, 13 G3831A A852T 4831D05 Hom 12, 13 G3585A V770M 12.70.00 4547H07 Het 12, 13 C3601T S775F 19.0 0.00 1427A06 Hom 12, 13 C3627TR784C 4755H09 Het 12, 13 G3628A R784H 4752C09 Hom 12, 13 G3643A W789*4544B07 Het 12, 13 G3644A W789* 2335D03 Hom 12, 13 C3655T P793L 4548B05Hom 12, 13 G3666A E797K 2197G10 Het

In one embodiment, the disclosure relates to multiple non-transgenicmutations in the Lpx promoter of the D genome including but not limitedto 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations. In oneembodiment, one or more non-transgenic mutations are in both alleles ofthe Lpx-D1 promoter in the D genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the Lpx-D1promoter of the D genome.

In one embodiment, one or more mutations are in the Lpx-D1 promoter ofthe D genome. In one embodiment, the disclosure relates to multiplenon-transgenic mutations in the Lpx-D1 promoter including but notlimited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

Table 3 provides representative examples of mutations created andidentified in Lpx-D1 promoter in the D genome of wheat plants, varietyExpress. Nucleotide changes are identified according to their positionsin SEQ ID NO: 15. Zygosity refers to whether the mutation isheterozygous (Het) or homozygous (Hom) in the M2 plant.

TABLE 3 Representative mutations in the Lpx-D1 promoter in the D genomeNucleotide Mutation Primer (SEQ ID SEQ IDs NO: 15) Description Zygosity16, 17 C1275T 1687E12 Het 16, 17 C1276T 1706B01 Hom 16, 17 C1292T1686C07 Hom 16, 17 C1292T 1686C07 Hom 16, 17 C1339T 1705B01 Het 16, 17G1349A 1686F08 Het 16, 17 C1411T 1687F07 Het 16, 17 C1418T 1685F01 Hom16, 17 G1538A 1691D07 Hom 16, 17 G1546A 1705A09 Het 16, 17 G1602A1691F03 Het 16, 17 G1736A 1685E01 Hom 16, 17 C1752T 1705H10 Hom 16, 17C1834T 1686B02 Hom 16, 17 C1857T 1705H08 Hom 16, 17 G1907A 1684F07 Hom16, 17 G1935A 1691D05 Het 16, 17 C1276T 1738F01 Hom 16, 17 C1282T1752H03 Hom 16, 17 C1333T 1743B10 Hom 16, 17 G1407A 2162C07 Het 16, 17G1421A 1738B02 Het 16, 17 G1474A 2163B03 Het 16, 17 C1485T 1750C09 Het16, 17 C1502T 2162C05 Het 16, 17 G1547A 2163B05 Het 16, 17 G1558A1751C10 Het 16, 17 C1625T 1741D12 Het 16, 17 C1627T 1737B09 Hom 16, 17C1655T 1737H08 Het 16, 17 C1752T 1737G07 Hom 16, 17 C1792T 1743G08 Hom16, 17 G1795A 1738E08 Het 16, 17 G1839A 1737G06 Hom 16, 17 G1846A1741G03 Het 16, 17 G1897A 1741F06 Het 16, 17 G1907A 1750H06 Het 16, 17G1940A 1741F07 Het 16, 17 C2102T 1752G03 Het 16, 17 C1238T 2164C02 Het16, 17 C1276T 2168C09 Het 16, 17 C1277T 2168D09 Het 16, 17 G1352A2171C12 Het 16, 17 C1414T 2169C01 Het 16, 17 G1518A 2171F05 Hom 16, 17G1532A 2167E08 Het 16, 17 C1565T 2168B09 Het 16, 17 G1697A 2168E07 Het16, 17 C1715T 2166A12 Het 16, 17 C1779T 2166F08 Het 16, 17 C1814T2171E05 Het 16, 17 G1858A 2168D11 Het 16, 17 G1871A 2168E03 Het 16, 17C1888T 2166E01 Het 16, 17 G1911A 2163A08 Het 16, 17 G1939A 2166B08 Het16, 17 G1269A 2176H02 Het 16, 17 G1269A 2176F08 Het 16, 17 G1291A2176D11 Hom 16, 17 G1297A 2176G02 Hom 16, 17 C1315T, C1569T 2190B11 Het16, 17 C1484T 2190C05 Hom 16, 17 C1504T 2189C02 Het 16, 17 G1512A2172H08 Het 16, 17 G1518A 2171H10 Het 16, 17 G1546A 2176G08 Hom 16, 17C1557T 2189H06 Het 16, 17 C1568T 2172E03 Hom 16, 17 G1634A 2191E06 Het16, 17 C1695T 2189B06 Het 16, 17 C1709T 2178B11 Het 16, 17 C1715T2178E01 Het 16, 17 C1725T 2176C04 Hom 16, 17 G1759A 2175C11 Het 16, 17C1785T 2175F08 Het 16, 17 G1824A 2190D02 Het 16, 17 G1844A 2189G09 Het16, 17 C1121T 2196G02 Het 16, 17 C1279T 2196F05 Het 16, 17 C1312T2196A05 Hom 16, 17 G1382A 2195E02 Het 16, 17 C1484T 2194C07 Het 16, 17C1705T 2194F11 Het 16, 17 G1731A 2192A08 Het 16, 17 G1821A 2330B06 Het16, 17 G1822A, G1940A 2195D06 Het 16, 17 C1856T 2319A05 Het 16, 17G1887A 2195C03 Hom 16, 17 G1906A 2196B02 Het 16, 17 C1414T 4545F03 Hom16, 17 C1483T 4546E11 Het 16, 17 G1526A 4547G04 Het 16, 17 C1831T4543C05 Hom 16, 17 G1845A 4544A03 Hom 16, 17 G1966A 4547G09 Het

In one embodiment, the invention relates to a polynucleotide of the Lpx1gene in the D genome with one or more non-transgenic mutations listed inTable 2 and corresponding to SEQ ID NO: 1. In another embodiment, thepolynucleotide with one or more non-transgenic mutations listed in Table2 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater than 99% identical to SEQ ID NO: 1. In yet anotherembodiment, the polynucleotide with one or more non-transgenic mutationslisted in Table 2 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO: 1.

In still another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 2 codes for a Lpx-D1 protein,wherein the Lpx-D1 protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 3. Instill another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 2 codes for a Lpx-D1 protein,wherein the Lpx-D1 protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO: 3.

In still another embodiment, the invention relates to a polynucleotideof the Lpx-D1 promoter in the D genome with one or more non-transgenicmutations listed in Table 3 and corresponding to SEQ ID NO: 15. Inanother embodiment, the polynucleotide with one or more non-transgenicmutations listed in Table 3 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQID NO: 15. In yet another embodiment, the polynucleotide with one ormore non-transgenic mutations listed in Table 3 is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than99% similar to SEQ ID NO: 15.

B. Lpx1 Proteins

In yet another embodiment, the disclosure relates to one or morenon-transgenic mutations in the Lpx1 genes (as discussed above in thesection entitled Lpx1 Mutations) that result in an Lpx1 protein with oneor more mutations as compared to wild type Lpx1 protein. In oneembodiment, the non-transgenic mutations include but are not limited tothe mutations recited in Tables 1-3, corresponding mutations inhomoeologues, and combinations thereof.

In another embodiment, the disclosure relates to one or morenon-transgenic mutations in the Lpx1 gene or promoter that inhibitsproduction of the Lpx1 protein. In some embodiments, a mutation in theLpx1 gene or promoter reduces expression of the Lpx1 protein. In otherembodiments, a mutation in the Lpx1 gene or promoter creates an unstableor reduced function Lpx1 protein. In another embodiment, thenon-transgenic mutations include but are not limited to the mutationsrecited in Tables 1-3, corresponding mutations in homoeologues, andcombinations thereof.

1. Expression Level of Lpx1 Proteins

In another embodiment, the expression level of Lpx1 proteins with one ormore mutations disclosed herein is reduced to 0-2%, 2-5%, 5-7%, 7-10%,10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-60%,60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of the expression level ofthe wild type Lpx1 protein.

In yet another embodiment, the expression level of Lpx-D1 protein withone or more mutations disclosed herein is reduced to 0-2%, 2-5%, 5-7%,7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%,50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of the expressionlevel of the wild type Lpx-D1 protein.

In still another embodiment, the expression level of Lpx-B1.1a proteinwith one or more mutations disclosed herein is reduced to 0-2%, 2-5%,5-7%, 7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%,45-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of theexpression level of the wild type Lpx-B1.1a protein.

In still another embodiment, the expression level of Lpx-B1.1b proteinwith one or more mutations disclosed herein is reduced to 0-2%, 2-5%,5-7%, 7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%,45-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of theexpression level of the wild type Lpx-B1.1b protein.

In still another embodiment, the expression level of Lpx-B1.1c proteinwith one or more mutations disclosed herein is reduced to 0-2%, 2-5%,5-7%, 7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%,45-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of theexpression level of the wild type Lpx-B1.1c protein.

In still another embodiment, the expression level of Lpx-B1.2 proteinwith one or more mutations disclosed herein is reduced to 0-2%, 2-5%,5-7%, 7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%,45-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of theexpression level of the wild type Lpx-B1.2 protein.

In still another embodiment, the expression level of Lpx-B1.3 proteinwith one or more mutations disclosed herein is reduced to 0-2%, 2-5%,5-7%, 7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%,45-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of theexpression level of the wild type Lpx-B1.3 protein.

2. Activity of Lpx1 Proteins

In yet another embodiment, the lipoxygenase activity of the Lpx proteinwith one or more mutations disclosed herein is reduced to 0-1, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 86, 97, 98, 99% and by more than 99% of the activity level of thewild type Lpx1 protein. In another embodiment, the Lpx1 protein with oneor more mutations disclosed herein has no activity or zero activity ascompared to wild type Lpx1 protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinwith one or more mutations disclosed herein is from 1-10% or from 10-30%or from 30-50% or from 50-70% or from 70-80% or from 80-90% or from90-95% of the activity level of the wild type Lpx1 protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-D1 gene with one or more mutations disclosed herein isreduced to 0-1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 86, 97, 98, 99% and by more than 99% of theactivity level of the wild type Lpx-D1 protein. In another embodiment,the Lpx-D1 protein with one or more mutations disclosed herein has noactivity or zero activity as compared to wild type Lpx-D1 protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-D1 gene with one or more mutations disclosed herein is from1-10% or from 10-30% or from 30-50% or from 50-70% or from 70-80% orfrom 80-90% or from 90-95% of the activity level of the wild type Lpx-D1protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.1a gene with one or more mutations disclosed herein isreduced to 0-1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 86, 97, 98, 99% and by more than 99% of theactivity level of the wild type Lpx-B1.1a protein. In anotherembodiment, the Lpx-B1.1a protein with one or more mutations disclosedherein has no activity or zero activity as compared to wild typeLpx-B1.1a protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.1a gene with one or more mutations disclosed herein isfrom 1-10% or from 10-30% or from 30-50% or from 50-70% or from 70-80%or from 80-90% or from 90-95% of the activity level of the wild typeLpx-B1.1a protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.1b gene with one or more mutations disclosed herein isreduced to 0-1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 86, 97, 98, 99% and by more than 99% of theactivity level of the wild type Lpx1 protein. In another embodiment, theLpx-B1.1b protein with one or more mutations disclosed herein has noactivity or zero activity as compared to wild type Lpx-B1.1b protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.1b gene with one or more mutations disclosed herein isfrom 1-10% or from 10-30% or from 30-50% or from 50-70% or from 70-80%or from 80-90% or from 90-95% of the activity level of the wild typeLpx-B1.1b protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.1c gene with one or more mutations disclosed herein isreduced to 0-1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 86, 97, 98, 99% and by more than 99% of theactivity level of the wild type Lpx-B1.1c protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.1c gene with one or more mutations disclosed herein isfrom 1-10% or from 10-30% or from 30-50% or from 50-70% or from 70-80%or from 80-90% or from 90-95% of the activity level of the wild typeLpx-B1.1c protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.2 gene with one or more mutations disclosed herein isreduced to 0-1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 86, 97, 98, 99% and by more than 99% of theactivity level of the wild type Lpx-B1.2 protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.2 gene with one or more mutations disclosed herein isfrom 1-10% or from 10-30% or from 30-50% or from 50-70% or from 70-80%or from 80-90% or from 90-95% of the activity level of the wild typeLpx-B1.2 protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinfrom the Lpx-B1.3 gene with one or more mutations disclosed herein isreduced to 0-1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 86, 97, 98, 99% and by more than 99% of theactivity level of the wild type Lpx-B1.3 protein.

In yet another embodiment, the lipoxygenase activity of the Lpx1 proteinproduced from the Lpx-B1.3 gene with one or more mutations disclosedherein is from 1-10% or from 10-30% or from 30-50% or from 50-70% orfrom 70-80% or from 80-90% or from 90-95% of the activity level of thewild type Lpx-B1.3 protein.

C. Oxidative Stability/Increased Shelf Life

In yet another embodiment, the disclosure relates to one or morenon-transgenic mutations in an Lpx1 gene (as discussed above in thesection entitled Lpx1 Mutations) that results in increased shelf life.

In yet another embodiment, the disclosure relates to one or morenon-transgenic mutations in an Lpx1 gene (as discussed above in thesection entitled Lpx1 Mutations) that results in increased oxidativestability of flour made from wheat grains with one or more Lpx1mutations as compared to wild type wheat grains. In one embodiment, thenon-transgenic mutations include but are not limited to the mutationsrecited in Tables 1-3, corresponding mutations in homoeologues, andcombinations thereof.

In yet another embodiment, the shelf life of whole grain flour made fromwheat grains with one or more Lpx1 mutations disclosed herein isincreased from the typical shelf life of whole grain flour. Millerscommonly stamp ‘use by’ dates of 3-9 months after milling for wholegrain flour in the United States, but this shelf life can be reduced to1-3 months by high storage temperatures and humidity. Shelf life can bedetermined by sensory characteristics of the flour and products madefrom it including color, flavor, texture, aroma, performance or overallpreference of the finished product. Trained panelists can be used toassess differences between materials.

In yet another embodiment, shelf life of whole grain flour made fromwheat grain with one or more mutations disclosed herein is increased by1-9 months, or 2-10 months, or 3-11 months, or 4-12 months, or 5-13months, or 6-14 months, or 7-15 months, or 8-16 months, or 9-17 months,or 10-18 months or 11-19 months, or 12-20 months, or 13-21 months, or14-22 months, or 15-23 or 16-24 months as compared to the shelf life ofwhole grain flour made from wild-type grain.

In yet another embodiment, shelf life of whole grain flour made fromwheat grain with one or more mutations disclosed herein is increased by1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months,15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months, 24 months, 25 months, 26 months, 27months, 28 months, 29 months, 30 months, or greater than 30 months ascompared to the shelf life of whole grain flour made from wild-typegrain.

In yet another embodiment, the oxidative stability of whole grain flourmade from wheat grain with one or more mutations disclosed herein isincreased due to the decreased production of the decomposition productsof fatty acids that can affect the smell or flavor of the product. Notto be bound by any particular theory, the oxidative stability of wholegrain flour made from wheat grain with one or more mutations disclosedherein is increased due to the decreased production of the decompositionproducts of fatty acids.

In yet another embodiment, the production of decomposition products offatty acids, including but not limited to hexanal, or nonenal, ortrihydroxydecanoic acid, among others is decreased in whole grain flourmade from wheat grain with one or more mutations disclosed herein.

In still another embodiment, the production of decomposition products offatty acids, including but not limited to hexanal, or nonenal, ortrihydroxydecanoic acid, is decreased in whole grain flour made fromwheat grain with one or more mutations disclosed herein by 0-1, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 86, 97, 98, 99% and by more than 99% as compared to the productionof degradation products of fatty acids in whole grain flour made fromthe wild-type grain.

In another embodiment, the production of decomposition products of fattyacids, including but not limited to hexanal, or nonenal, ortrihydroxydecanoic acid, is decreased in whole grain flour made fromwheat grain with one or more mutations disclosed herein from 1% to 5%,or from 5% to 10%, or from 10% to 15%, or from 15% to 20%, or from 20%to 25%, or from 25% to 30%, or from 30% to 35%, or from 35% to 40%, orfrom 40% to 45%, or from 45% to 50%, or from 50% to 55%, or from 55% to60%, or from 65% to 70%, or from 70% to 75%, or from 75% to 80%, or from80% to 85%, or from 85% to 90%, or from 90% to 95%, or from 95% to 99%,or by more than 99% as compared to the production of degradationproducts of fatty acids in whole grain flour made from the wild-typegrain.

In another embodiment, the production of decomposition products of fattyacids, including but not limited to hexanal, or nonenal, ortrihydroxydecanoic acid, is decreased in whole grain flour made fromwheat grain with one or more mutations disclosed herein by at least 5%,or at least 10%, or at least 15%, or at least 20%, or at least 25%, orat least 30%, or at least 35%, or at least 40%, or at least 45%, or atleast 50%, or at least 55%, or at least 60%, or at least 65%, or atleast 70%, or at least 75%, or at least 80%, or at least 85%, or atleast 90%, or at least 95% as compared to the production of degradationproducts of fatty acids in whole grain flour made from the wild-typegrain.

In another embodiment, the production of trihydroxydecanoic acid(pinellic acid) is decreased in whole grain flour or dough or bread orother products made from wheat grain with one or more mutationsdisclosed herein from 1% to 5%, or from 5% to 10%, or from 10% to 15%,or from 15% to 20%, or from 20% to 25%, or from 25% to 30%, or from 30%to 35%, or from 35% to 40%, or from 40% to 45%, or from 45% to 50%, orfrom 50% to 55%, or from 55% to 60%, or from 65% to 70%, or from 70% to75%, or from 75% to 80%, or from 80% to 85%, or from 85% to 90%, or from90% to 95%, or from 95% to 99%, or by more than 99% as compared to theproduction of pinellic acid in whole grain flour made from the wild-typegrain.

In another embodiment, the production of trihydroxydecanoic acid(pinellic acid) is decreased in whole grain flour or dough or bread orother products made from wheat grain with one or more mutationsdisclosed herein by at least 5%, or at least 10%, or at least 15%, or atleast 20%, or at least 25%, or at least 30%, or at least 35%, or atleast 40%, or at least 45%, or at least 50%, or at least 55%, or atleast 60%, or at least 65%, or at least 70%, or at least 75%, or atleast 80%, or at least 85%, or at least 90%, or at least 95% as comparedto the production of pinellic acid in whole grain flour made from thewild-type grain.

III. Transgenes

In one embodiment, the disclosure relates to a transgenic plant thatcomprises a transgene that encodes a polynucleotide, whichdown-regulates the expression of the Lpx1 gene and/or the activity ofthe Lpx1 protein. Examples of such polynucleotides include, but are notlimited to, antisense polynucleotide, a sense polynucleotide, acatalytic polynucleotide, an artificial microRNA or a duplex RNAmolecule.

In one embodiment, the disclosure relates to a wheat plant comprising atransgene that reduces the expression of the Lpx1 gene and/or activityof the Lpx1 protein, wherein grain from the wheat plant has increasedoxidative stability or increased shelf-life as compared to grains from awild type plant.

A. Antisense Polynucleotides

The term “antisense polynucleotide” shall be taken to refer to a DNA orRNA, or combination thereof, molecule that is complementary to at leasta portion of a specific mRNA molecule encoding Lpx1 and capable ofinterfering with a post-transcriptional event such as mRNA translation.

An antisense polynucleotide in a plant will hybridize to a targetpolynucleotide under physiological conditions. As used herein, the term“an antisense polynucleotide which hybridizes under physiologicalconditions” means that the polynucleotide (which is fully or partiallysingle stranded) is at least capable of forming a double strandedpolynucleotide with mRNA encoding a protein.

Antisense molecules may include sequences that correspond to thestructural gene or for sequences that effect control over the geneexpression or splicing event. For example, the antisense sequence maycorrespond to the targeted coding region of Lpx1 or the 5′-untranslatedregion (UTR) or the 3′-UTR or combination of these. It may becomplementary in part to intron sequences, which may be spliced outduring or after transcription, preferably only to exon sequences of thetarget gene. In view of the generally greater divergence of the UTRs,targeting these regions provides greater specificity of gene inhibition.

The length of the antisense sequence should be at least 19 contiguousnucleotides, preferably at least 50 nucleotides, and more preferably atleast 100, 200, 500 or 1000 nucleotides. The full-length sequencecomplementary to the entire gene transcript may be used. The length ismost preferably 100-2000 nucleotides. The degree of identity of theantisense sequence to the targeted transcript should be at least 90% andmore preferably 95-100%. The antisense RNA molecule may of coursecomprise unrelated sequences, which may function to stabilize themolecule.

B. Catalytic Polynucleotides

The term catalytic polynucleotide/nucleic acid refers to a DNA moleculeor DNA-containing molecule (also known in the art as a “deoxyribozyme”)or an RNA or RNA-containing molecule (also known as a “ribozyme”) whichspecifically recognizes a distinct substrate and catalyzes the chemicalmodification of this substrate. The nucleic acid bases in the catalyticnucleic acid can be bases A, C, G, T (and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence forspecific recognition of a target nucleic acid, and a nucleic acidcleaving enzymatic activity (also referred to herein as the “catalyticdomain”).

The ribozymes in plants disclosed herein and DNA encoding the ribozymescan be chemically synthesized using methods well known in the art. Theribozymes can also be prepared from a DNA molecule (that upontranscription, yields an RNA molecule) operably linked to an RNApolymerase promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNApolymerase. When the vector also contains an RNA polymerase promoteroperably linked to the DNA molecule, the ribozyme can be produced invitro upon incubation with RNA polymerase and nucleotides. In a separateembodiment, the DNA can be inserted into an expression cassette ortranscription cassette. After synthesis, the RNA molecule can bemodified by ligation to a DNA molecule having the ability to stabilizethe ribozyme and make it resistant to RNase.

As with antisense polynucleotides described herein, the catalyticpolynucleotides should also be capable of hybridizing a target nucleicacid molecule (for example mRNA encoding LPX1) under “physiologicalconditions,” namely those conditions within a plant cell.

C. RNA Interference

RNA interference (RNAi) is particularly useful for specificallyinhibiting the production of a particular protein. This technologyrelies on the presence of dsRNA molecules that contain a sequence thatis essentially identical to the mRNA of the gene of interest or partthereof. Conveniently, the dsRNA can be produced from a single promoterin a recombinant vector or host cell, where the sense and anti-sensesequences are flanked by an unrelated sequence which enables the senseand anti-sense sequences to hybridize to form the dsRNA molecule withthe unrelated sequence forming a loop structure. The design andproduction of suitable dsRNA molecules for the present invention is wellwithin the capacity of a person skilled in the art, particularlyconsidering, WO 99/32619, WO 99/53050, WO 99/49029, and WO 01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded (duplex) RNA product(s) with homology tothe target gene to be inactivated. The DNA therefore comprises bothsense and antisense sequences that, when transcribed into RNA, canhybridize to form the double-stranded RNA region. In a preferredembodiment, the sense and antisense sequences are separated by a spacerregion that comprises an intron which, when transcribed into RNA, isspliced out. This arrangement has been shown to result in a higherefficiency of gene silencing. The double-stranded region may compriseone or two RNA molecules, transcribed from either one DNA region or two.The presence of the double stranded molecule is thought to trigger aresponse from an endogenous plant system that destroys both the doublestranded RNA and also the homologous RNA transcript from the targetplant gene, efficiently reducing or eliminating the activity of thetarget gene.

The length of the sense and antisense sequences that hybridize shouldeach be at least 19 contiguous nucleotides, preferably at least 30 or 50nucleotides, and more preferably at least 100, 200, 500 or 1000nucleotides. The full-length sequence corresponding to the entire genetranscript may be used. The lengths are most preferably 100-2000nucleotides. The degree of identity of the sense and antisense sequencesto the targeted transcript should be at least 85%, preferably at least90% and more preferably 95-100%. The RNA molecule may of course compriseunrelated sequences which may function to stabilize the molecule. TheRNA molecule may be expressed under the control of a RNA polymerase IIor RNA polymerase III promoter. Examples of the latter include tRNA orsnRNA promoters.

In one embodiment, small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. Preferably, the target mRNA sequencecommences with the dinucleotide AA, comprises a GC-content of about30-70% (preferably, 30-60%, more preferably 40-60% and more preferablyabout 45%-55%), and does not have a high percentage identity to anynucleotide sequence other than the target in the genome of the barleyplant in which it is to be introduced, e.g., as determined by standardBLAST search.

D. microRNA

MicroRNA regulation is a clearly specialized branch of the RNA silencingpathway that evolved towards gene regulation, diverging fromconventional RNAi/Post transcriptional Gene Silencing (PTGS). MicroRNAsare a specific class of small RNAs that are encoded in gene-likeelements organized in a characteristic inverted repeat. Whentranscribed, microRNA genes give rise to stem-looped precursor RNAs fromwhich the microRNAs are subsequently processed. MicroRNAs are typicallyabout 21 nucleotides in length. The released miRNAs are incorporatedinto RISC-like complexes containing a particular subset of Argonauteproteins that exert sequence-specific gene repression.

E. Co-suppression

Another molecular biological approach that may be used isco-suppression. The mechanism of co-suppression is not well understoodbut is thought to involve post-transcriptional gene silencing (PTGS) andin that regard may be very similar to many examples of antisensesuppression. It involves introducing an extra copy of a gene or afragment thereof into a plant in the sense orientation with respect to apromoter for its expression. The size of the sense fragment, itscorrespondence to target gene regions, and its degree of sequenceidentity to the target gene are as for the antisense sequences describedabove. In some instances the additional copy of the gene sequenceinterferes with the expression of the target plant gene. Reference ismade to WO 97/20936 and EP 0465572 for methods of implementingco-suppression approaches.

IV. Genomic Editing

In one embodiment, the disclosure relates to a plant with reducedexpression of the Lpx1 gene and/or reduced activity of the Lpx1 protein,wherein reduced expression of the Lpx1 gene and/or reduced activity ofthe Lpx1 protein is achieved by genomic editing.

In one embodiment, the disclosure relates to a wheat plant with agenomically edited Lpx1 gene, wherein grain from the wheat plant hasincrease oxidative stability or increased shelf life as compared tograins from a wild type plant.

Genome editing, or genome editing with engineered nucleases (GEEN), is atype of genetic engineering in which DNA is inserted, replaced, orremoved from a genome using artificially engineered nucleases, or“molecular scissors.” The nucleases create specific double-strandedbreaks (DSBs) at desired locations in the genome, and harness the cell'sendogenous mechanisms to repair the induced break by natural processesof homologous recombination (HR) and nonhomologous end-joining (NHEJ).There are currently four main families of engineered nucleases beingused: Zinc finger nucleases (ZFNs), Transcription Activator-LikeEffector Nucleases (TALENs), the CRISPR/Cas system, and engineeredmeganuclease with a re-engineered homing endonucleases.

A. Zinc Finger Nucleases (ZFNs)

Zinc-finger nucleases (ZFNs) are artificial restriction enzymesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain. Zinc finger domains can be engineered to target specific desiredDNA sequences and this enables zinc-finger nucleases to target uniquesequences within complex genomes. By taking advantage of endogenous DNArepair machinery, these reagents can be used to precisely alter thegenomes of higher organisms.

ZFNs consist of an engineered zinc finger DNA-binding domain fused tothe cleavage domain of the Fold restriction endonuclease. ZFNs can beused to induce double-stranded breaks (DSBs) in specific DNA sequencesand thereby promote site-specific homologous recombination with anexogenous template. The exogenous template contains the sequence that isto be introduced into the genome.

Publicly available methods for engineering zinc finger domains include:(1) Context-dependent Assembly (CoDA), (2) Oligomerized Pool Engineering(OPEN), and (3) Modular Assembly.

In one embodiment, the disclosure relates to reducing expression of theLpx1 gene and/or reducing activity of the Lpx1 protein using ZFNs.

B. Transcription Activator-Like Effector Nucleases (TALENs)

TALEN is a sequence-specific endonuclease that consists of atranscription activator-like effector (TALE) and a FokI endonuclease.TALE is a DNA-binding protein that has a highly conserved central regionwith tandem repeat units of 34 amino acids. The base preference for eachrepeat unit is determined by two amino acid residues called therepeat-variable di-residue (RVD), which recognizes one specificnucleotide in the target DNA. Arrays of DNA-binding repeat units can becustomized for targeting specific DNA sequences. As with ZFNs,dimerization of two TALENs on targeted specific sequences in a genomeresults in FokI-dependent introduction of DSBs, stimulating homologydirected repair (HDR) and Non-homologous end joining (NHEJ) repairmechanisms.

In one embodiment, the disclosure relates to reducing expression of theLpx1 gene and/or reducing activity of the Lpx1 protein using TALENs.

C. CRISPR/Cas System

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)Type II system is an RNA-Guided Endonuclease technology for genomeengineering. There are two distinct components to this system: (1) aguide RNA and (2) an endonuclease, in this case the CRISPR associated(Cas) nuclease, Cas9.

The guide RNA is a combination of the endogenous bacterial crRNA andtracrRNA into a single chimeric guide RNA (gRNA) transcript. The gRNAcombines the targeting specificity of the crRNA with the scaffoldingproperties of the tracrRNA into a single transcript. When the gRNA andthe Cas9 are expressed in the cell, the genomic target sequence can bemodified or permanently disrupted.

The gRNA/Cas9 complex is recruited to the target sequence by thebase-pairing between the gRNA sequence and the complementarity to thetarget sequence in the genomic DNA. For successful binding of Cas9, thegenomic target sequence must also contain the correct ProtospacerAdjacent Motif (PAM) sequence immediately following the target sequence.The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomictarget sequence so that the wild-type Cas9 can cut both strands of DNAcausing a Double Strand Break (DSB). Cas9 will cut 34 nucleotidesupstream of the PAM sequence. A DSB can be repaired through one of twogeneral repair pathways: (I) NHEJ DNA repair pathway or (2) the HDRpathway. The NHEJ repair pathway often results in insertions/deletions(InDels) at the DSB site that can lead to frameshifts and/or prematurestop codons, effectively disrupting the open reading frame (ORF) of thetargeted gene.

The HDR pathway requires the presence of a repair template, which isused to fix the DSB. HDR faithfully copies the sequence of the repairtemplate to the cut target sequence. Specific nucleotide changes can beintroduced into a targeted gene by the use of HDR with a repairtemplate.

In one embodiment, the disclosure relates to reducing expression of theLpx1 gene and/or reducing activity of the Lpx1 protein using theCRISPR/cas9 system.

D. Meganuclease with Re-Engineered Homing Nuclease

Meganucleases are endodeoxyribonucleases characterized by a largerecognition site (double-stranded DNA sequences of 12 to 40 base pairs);as a result this site generally occurs only once in any given genome.For example, the 18-base pair sequence recognized by the I-SceImeganuclease would on average require a genome twenty times the size ofthe human genome to be found once by chance (although sequences with asingle mismatch occur about three times per human-sized genome).Meganucleases are therefore considered to be the most specific naturallyoccurring restriction enzymes.

Among meganucleases, the LAGLIDADG family of homing endonucleases hasbecome a valuable tool for the study of genomes and genome engineeringover the past fifteen years. By modifying their recognition sequencethrough protein engineering, the targeted sequence can be changed.

In one embodiment, the disclosure relates to reducing expression of theLpx1 gene and/or reducing activity of the Lpx1 protein using ameganuclease with a re-engineered homing nuclease.

V. Wheat Cultivars

In one embodiment, a wheat cultivar having at least one Lpx gene that isdiploid, tetraploid, or hexaploid may be used. In another embodiment,the wheat is Triticum aestivum.

In one embodiment, any cultivar of wheat can be used to create mutationsin an Lpx gene. In one embodiment, any cultivar of wheat can be used tocreate mutations in an Lpx-D1 gene. In another embodiment, any cultivarof wheat can be used to create mutations in an Lpx-B1.1a gene. Inanother embodiment, any cultivar of wheat can be used to createmutations in an Lpx-B1.1b gene. In another embodiment, any cultivar ofwheat can be used to create mutations in an Lpx-B1.1c gene. In anotherembodiment, any cultivar of wheat can be used to create mutations in anLpx-B1.2 gene. In another embodiment, any cultivar of wheat can be usedto create mutations in an Lpx-B1.3 gene. In another embodiment, anycultivar of wheat can be used to create mutations in an Lpx-A1 gene.

In one embodiment, any cultivar of wheat can be used as lines to crossLpx mutations into different cultivars.

In another embodiment, any cultivar of wheat having at least one Lpxgene may be used including but not limited to hard red spring wheat,hard white winter wheat, durum wheat, soft white spring wheat, softwhite winter wheat, hard red winter wheat, common wheat, spelt wheat,emmer wheat, pasta wheat and turgidum wheat.

In one embodiment, hard red spring wheat includes but is not limited toBullseye, Cabernet, Cal Rojo, Hank, Joaquin, Kelse, Lariat, Lassik,Malbec, Mika, PR 1404, Redwing, Summit 515, SY 314, Triple IV, Ultra,WB-Patron, WB-Rockland, Yecora Rojo, Accord, Aim, Anza, Baker, BethHashita, Bonus, Borah, Brim, Brooks, Buck Pronto, Butte 86, Cavalier,Challenger, Chief, Ciano T79, Colusa, Companion, Copper, Cuyama, Dash12, Eldon, Enano, Express, Expresso, Jefferson, Genero F81, Grandin,Helena 554, Hollis, Imuris T79, Inia 66R, Jerome, Kern, Len, Marshall,McKay, Nomad, Northwest 10, Oslo, Pavon F76, Pegasus, Pitic 62, PocoRed, Powell, Probrand 711, Probrand 751, Probrand 771, Probrand 775,Probred, Prointa Queguay, Prointa Quintal, Rich, RSI 5, Sagittario,Scarlet, Serra, Shasta, Solano, Spillman, Sprite, Stander, Stellar,Stoa, Success, Summit, Sunstar 2, Sunstar King, Tadinia, Tammy, Tanori71, Tara 2000, Tempo, Tesia T79, Topic, UI Winchester, Vance, Vandal,W444, Wampum, Wared, WB-Fuzion, Westbred 906R, Westbred 911, Westbred926, Westbred 936, Westbred Discovery, Westbred Rambo, Yolo, and Zeke.

In another embodiment, hard white wheat includes but is not limited toBlanca Fuerte, Blanca Grande 515, Blanca Royale, Clear White, Patwin,Patwin 515, WB-Cristallo, WB-Paloma, WB-Perla, Alta Blanca, BlancaGrande, Delano, Golden Spike, ID377S, Klasic, Lochsa, Lolo, Macon, Otis,Phoenix, Pima 77, Plata, Pristine, Ramona 50, Siete Cerros 66, Vaiolet,and Winsome.

In yet another embodiment, durum wheat includes but is not limited toCrown, Desert King, Desert King HP, Duraking, Fortissimo, Havasu,Kronos, Maestrale, Normanno, Orita, Platinum, Q-Max, RSI 59, Saragolla,Tango, Tipai, Topper, Utopia, Volante, WB-Mead, Westmore, Aldente,Aldura, Altar 84, Aruba, Bittern, Bravadur, Candura, Cortez, Deluxe,Desert Titan, Durex, Durfort, Eddie, Germains 5003D, Imperial, Kofa,Levante, Matt, Mead, Mexicali 75, Minos, Modoc, Mohawk, Nudura,Ocotillo, Produra, Reva, Ria, Septre, Sky, Tacna, Titan, Trump, Ward,Westbred 803, Westbred 881, Westbred 883, Westbred 1000D, WestbredLaker, Westbred Turbo, and Yavaros 79.

In another embodiment, soft white spring wheat includes but is notlimited to Alpowa, Alturas, Babe, Diva, JD, New Dirkwin, Nick, Twin,Whit, Blanca, Bliss, Calorwa, Centennial, Challis, Dirkwin, Eden,Edwall, Fielder, Fieldwin, Jubilee, Louise, Owens, Penawawa, Pomerelle,Sterling, Sunstar Promise, Super Dirkwin, Treasure, UI Cataldo, UIPettit, Urquie, Vanna, Waduel, Waduel 94, Wakanz, Walladay, Wawawai,Whitebird, and Zak.

In still another embodiment, soft white winter wheat includes but is notlimited to AP Badger, AP Legacy, Brundage 96, Bruneau, Cara, Goetze,Legion, Mary, Skiles, Stephens, SY Ovation, Tubbs, WB-Junction, WB-528,Xerpha, Yamhill, Barbee, Basin, Bitterroot, Bruehl, Castan, Chukar,Coda, Daws, Edwin, Eltan, Faro, Finch, Foote, Gene, Hill 81, Hiller,Hubbard, Hyak, Hyslop, Idaho 587, Kmor, Lambert, Lewjain, MacVicar,Madsen, Malcolm, Masami, McDermid, Moro, Nugaines, ORCF-101, ORCF-102,ORCF-103, Rod, Rohde, Rulo, Simon, Salute, Temple, Tres, Tubbs 06,UICF-Brundage, WB-523, and Weatherford.

In another embodiment, hard red winter wheat includes but is not limitedto Andrews, Archer, Batum, Blizzard, Bonneville, Boundary, Declo,Deloris, Finley, Garland, Hatton, Hoff, Longhorn, Manning, Meridian,Promontory, Vona, Wanser, Winridge.

In another embodiment, common wheat (hexaploid, free threshing),Triticum aestivum ssp aestivum includes but is not limited to Sonora,Wit Wolkoring, Chiddam Blanc De Mars, India-Jammu, Foisy.

In still another embodiment, spelt wheat (hexaploid, not freethreshing), Triticum aestivum ssp spelta includes but is not limited toSpanish Spelt, Swiss Spelt.

In yet another embodiment, Emmer Wheat (tetraploid), Triticum turgidumssp. dicoccum includes but is not limited to Ethiopian Blue Tinge.

In another embodiment, pasta wheat (tetraploid, free threshing),Triticum turgidum ssp durum includes but is not limited to Blue Beard,Durum-Iraq.

In yet another embodiment, Turgidum Wheat (tetraploid, free threshing),Triticum turgidum ssp turgidum includes but is not limited to Akmolinka,Maparcha.

In one embodiment, a cultivar of wheat having at least one Lpx1 genewith substantial percent identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO. 15 may be used with the methods andcompositions disclosed herein.

As used herein with regard to the wheat cultivars, “substantial percentidentity” means that the DNA sequence of the gene is sufficientlysimilar to SEQ ID NO: 1, 2, 4, and 5 at the nucleotide level to code fora substantially similar protein, allowing for allelic differences (oralternate mRNA splicing) between cultivars. In accordance with oneembodiment of the invention, “substantial percent identity” may bepresent when the percent identity in the coding region between the Lpx1gene and SEQ ID NO: 1, 2, 4, and 5 is as low as about 85%, provided thatthe percent identity in the conserved regions of the gene is higher(e.g., at least about 90%). Preferably the percent identity in thecoding region is 85-90%, more preferably 90-95%, and optimally, it isabove 95%. Thus, one of skill in the art may prefer to utilize a wheatcultivar having commercial popularity or one having specific desiredcharacteristics in which to create the Lpx1-mutated wheat plants,without deviating from the scope and intent of the present invention.Alternatively, one of skill in the art may prefer to utilize a wheatcultivar having few polymorphisms, such as an in-bred cultivar, in orderto facilitate screening for mutations within one or more Lpx1 genes inaccordance with the present invention.

VI. Representative Methodology for Identification of Lpx1 Mutations

In order to create and identify the Lpx1 mutations and wheat plantsdisclosed herein, a method known as TILLING (Targeting Induced LocalLesions IN Genomes) was utilized. See McCallum et al., NatureBiotechnology 18:455-457, 2000; McCallum et al., Plant Physiology,123:439-442, 2000; U.S. Publication No. 20040053236; and U.S. Pat. No.5,994,075, all of which are incorporated herein by reference. In thebasic TILLING methodology, plant materials, such as seeds, are subjectedto chemical mutagenesis, which creates a series of mutations within thegenomes of the seeds' cells. The mutagenized seeds are grown into adultM1 plants and self-pollinated. DNA samples from the resulting M2 plantsare pooled and are then screened for mutations in a gene of interest.Once a mutation is identified in a gene of interest, the seeds of the M2plant carrying that mutation are grown into adult M3 plants and screenedfor the phenotypic characteristics associated with the gene of interest.

The hexaploid cultivar Express was used.

In one embodiment, seeds from wheat are mutagenized and then grown intoM1 plants. The M1 plants are then allowed to self-pollinate and seedsfrom the M1 plant are grown into M2 plants, which are then screened formutations in their Lpx1 loci. While M1 plants can be screened formutations in accordance with alternative embodiments of the invention,one advantage of screening the M2 plants is that all somatic mutationscorrespond to germline mutations.

One of skill in the art will understand that a variety of wheat plantmaterials, including, but not limited to, seeds, pollen, plant tissue orplant cells, may be mutagenized in order to create the Lpx-mutated wheatplants disclosed herein. However, the type of plant material mutagenizedmay affect when the plant DNA is screened for mutations. For example,when pollen is subjected to mutagenesis prior to pollination of anon-mutagenized plant, the seeds resulting from that pollination aregrown into M1 plants. Every cell of the M1 plants will contain mutationscreated in the pollen, thus these M1 plants may then be screened forLpx1 mutations instead of waiting until the M2 generation.

Mutagens that create primarily point mutations and deletions,insertions, transversions, and or transitions, such as chemical mutagensor radiation, may be used to create the mutations. Mutagens conformingwith the method of the invention include, but are not limited to, ethylmethanesulfonate (EMS), methylmethane sulfonate (MMS),N-ethyl-N-nitrosourea (ENU), triethylmelamine (TEM),N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil,cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan,nitrogen mustard, vincristine, dimethylnitrosamine,N-methyl-N′-nitro-Nitrosoguanidine (MNNG), nitrosoguanidine,2-aminopurine, 7, 12 dimethyl-benz(a)anthracene (DMBA), ethylene oxide,hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO),diepoxybutane (BEB), and the like),2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethypaminopropylamino] acridinedihydrochloride (ICR-170), formaldehyde, fast neutrons, and gammairradiation. Spontaneous mutations in a Lpx1 gene that may not have beendirectly caused by the mutagen can also be identified.

Other methods such as genome editing can also be used to alter thesequence of a target gene including its promoter to up or down regulateexpression or activity. These methods are known to those skilled in theart, and include CRISPR, Talens, Zinc finger nucleases and miRNA amongother methods. For example, see a review of these methods by Xiong etal. Horticulture Research 2: 15019, 2015.

Any suitable method of plant DNA preparation now known or hereafterdevised may be used to prepare the wheat plant DNA for Lpx1 mutationscreening. For example, see Chen & Ronald, Plant Molecular BiologyReporter 17:53-57, 1999; Stewart and Via, Bio Techniques 14:748-749,1993. Additionally, several commercial kits designed for this purposeare available, including kits from Qiagen (Valencia, Calif.) andQbiogene (Carlsbad, Calif.).

In one embodiment, prepared DNA from individual wheat plants are pooledin order to expedite screening for mutations in one or more Lpx1 genesof the entire population of plants originating from the mutagenizedplant tissue. The size of the pooled group may be dependent upon thesensitivity of the screening method used. Preferably, groups of two ormore individual wheat plants are pooled.

In another embodiment, after the DNA samples are pooled, the pools aresubjected to Lpx1 sequence-specific amplification techniques, such asPolymerase Chain Reaction (PCR). For a general overview of PCR, see PCRProtocols: A Guide to Methods and Applications (Innis, Gelfand, Sninsky,and White, eds.), Academic Press, San Diego, 1990.

Any primer specific to an Lpx1 locus or the sequences immediatelyadjacent to one of these loci may be utilized to amplify the Lpx1sequences within the pooled DNA sample. Preferably, the primer isdesigned to amplify the regions of the Lpx1 locus where useful mutationsare most likely to arise. Most preferably, the primer is designed todetect exonic regions of one or more Lpx1 genes. Additionally, it ispreferable for the primer to target known polymorphic sites to designgenome specific primers in order to ease screening for point mutationsin a particular genome.

In one embodiment, primers are designed based upon the Lpx-D1 andLpx-B1.2 DNA sequences (SEQ ID NOs: 1, 2, 4, 5 and 15). Exemplaryprimers (SEQ ID NOs: 7-14, 16 and 17) that have proven useful inidentifying useful mutations within the Lpx1 sequences are shown inTable 4. These primers are also detailed in the Sequence Listingappended hereto.

TABLE 4 Exemplary TILLING Primers SEQ ID NO: 7 TaLpx1D_4_L-1TGGTGAGAGCACGCAAATCTTACTTGG SEQ ID NO: 8 TaLpxB1.2-D1-5R1CGTTTCAATCATAGGTCAGTTGTGCATCGA SEQ ID NO: 9 TaLpx1D_67_R_3CGCGTACGGGTAGTCCGACACCAGAAG SEQ ID NO: 10 TaLpx1D_In1_L4GCATGCCATGGAAAGAAGAGACAATAGTAGC SEQ ID NO: 11 TaLpx1D_3_R-1TGCGTGCTCTCACCATGGACAACATACATA SEQ ID NO: 12 TaLpx1D_Ex5_L6GCAGGCGCTGGAAAGTAACAGGCTCT SEQ ID NO: 13 TaLpx1D_Ex7_R3TGGACGAGACGAAGCTCCGATGTACCA SEQ ID NO: 14 TaLpx1.2B_4_L-1GAGGTGAGAGCGTGCCTGATCTTAATTTG SEQ ID NO: 16 LpxD1proLTCATGCCGCTGATCGTCGC SEQ ID NO: 17 LpxD1proR CTTGCTGCTATTTCAGTACCG

In another embodiment, the PCR amplification products may be screenedfor Lpx1 mutations using any method that identifies nucleotidedifferences between wild type and mutant sequences. These may include,for example, without limitation, sequencing, denaturing high pressureliquid chromatography (dHPLC), constant denaturant capillaryelectrophoresis (CDCE), temperature gradient capillary electrophoresis(TGCE) (see Li et al., Electrophoresis 23(10):1499-1511, 2002), or byfragmentation using enzymatic cleavage, such as used in the highthroughput method described by Colbert et al., Plant Physiology126:480-484, 2001. Preferably, the PCR amplification products areincubated with an endonuclease that preferentially cleaves mismatches inheteroduplexes between wild type and mutant sequences.

In another embodiment, cleavage products are electrophoresed using anautomated sequencing gel apparatus, and gel images are analyzed with theaid of a standard commercial image-processing program.

In yet another embodiment, once an M2 plant having a mutated Lpx1 genesequence is identified, the mutations are analyzed to determine theireffect on the expression, translation, and/or activity of an Lpx1enzyme. In one embodiment, the PCR fragment containing the mutation issequenced, using standard sequencing techniques, in order to determinethe exact location of the mutation in relation to the overall Lpx1sequence. Each mutation is evaluated in order to predict its impact onprotein function (i.e., from completely tolerated to causingloss-of-function) using bioinformatics tools such as SIFT (SortingIntolerant from Tolerant; Ng and Henikoff, Nucleic Acids Research31:3812-3814, 2003), PSSM (Position-Specific Scoring Matrix; Henikoffand Henikoff, Computer Applications in the Biosciences 12:135-143, 1996)and PARSESNP (Taylor and Greene, Nucleic Acids Research 31:3808-3811,2003). For example, a SIFT score that is less than 0.05 and a largechange in PSSM score (e.g., roughly 10 or above) indicate a mutationthat is likely to have a deleterious effect on protein function. Theseprograms are known to be predictive, and it is understood by thoseskilled in the art that the predicted outcomes are not always accurate.

In another embodiment, if the initial assessment of a mutation in the M2plant indicates it to be of a useful nature and in a useful positionwithin an Lpx1 gene, including its promoter, then further phenotypicanalysis of the wheat plant containing that mutation may be pursued. Inhexaploid wheat, mutations in each of the A, B and D genomes usuallymust be combined before a phenotype can be detected. In tetraploidwheat, A and B genome mutations are combined. In addition, the mutationcontaining plant can be backcrossed or outcrossed two times or more inorder to eliminate background mutations at any generation. Then thebackcrossed or outcrossed plant can be self-pollinated or crossed inorder to create plants that are homozygous for the Lpx1 mutations.

Several physical characteristics of these homozygous Lpx1 mutant plantsare assessed to determine if the mutation results in a useful phenotypicchange in the wheat plant without resulting in undesirable negativeeffects, such as significantly reduced seed yields.

VII. Methods of Producing a Wheat Plant

In another embodiment, the disclosure relates to a method for producinga wheat plant with increased oxidative stability. In another embodiment,the invention relates to a method for producing a wheat plant with anincreased shelf-life.

In another embodiment, the disclosure relates to a method ofout-crossing Lpx1 gene mutations to wild type wheat.

In another embodiment, the disclosure relates to a method for producinga wheat plant having increased oxidative stability and products fromgrain of said wheat plant having increased shelf life. In still anotherembodiment, the invention relates to a method for producing a wheatplant having reduced activity of one or more Lpx1 enzymes compared tothe wild type wheat plants.

In one embodiment, the method comprises inducing at least onenon-transgenic mutation in at least one copy of an Lpx1 gene in plantmaterial or plant parts from a parent wheat plant; growing or using themutagenized plant material to produce progeny wheat plants; analyzingmutagenized plant material and/or progeny wheat plants to detect atleast one mutation in at least one copy of a Lpx1 gene; and selectingprogeny wheat plants that have at least one mutation in at least onecopy of an Lpx1 gene.

In another embodiment, the method further comprises crossing progenywheat plants that have at least one mutation in at least one copy of anLpx1 gene with other progeny wheat plants that have at least onemutation in a different copy of an Lpx1 gene. The process of identifyingprogeny wheat plants with mutations and crossing said progeny wheatplants with other progeny wheat plants, which have mutations, can berepeated to produce progeny wheat plants with reduced Lpx1 enzymeactivity.

In another embodiment, the level of activity of the Lpx1 protein in thewheat plant is reduced to 0-2%, 2-5%, 5-7%, 7-10%, 10-15%, 15-20%,20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-60%, 60-70%, 70-80%,80-90%, 90-95%, or 95-99% of the level of activity of the Lpx1 proteinin the wild type plant.

A. Methods of Producing a Wheat Plant with One or More Mutations in theLpx1 Gene in More than One Genome

In still another embodiment, the disclosure relates to a method forproducing a wheat plant comprising inducing at least one non-transgenicmutation in at least one copy of an Lpx1 gene in plant material from aparent wheat plant that comprises a mutation in an Lpx1 gene; growing orusing the mutagenized plant material to produce progeny wheat plants;and selecting progeny wheat plants that have at least one mutation in atleast two copies of an Lpx1 gene. For example, the parent wheat plantmay have a mutation in an Lpx-D1 gene of the D genome. The selectedprogeny wheat plants may have a mutation in an Lpx-D1 gene of the Dgenome and one or more mutations in the Lpx1 gene of the B genome. Thisexample is provided merely for clarification and should not limit themethods disclosed herein.

In yet another embodiment, the invention relates to a method forproducing a wheat plant comprising inducing at least one non-transgenicmutation in at least one copy of an Lpx1 gene in plant material from aparent wheat plant that comprises at least one mutation in two Lpx1genes; growing or using the mutagenized plant material to produceprogeny wheat plants; and selecting progeny wheat plants that have atleast one mutation in three copies of an Lpx1 gene. In this embodiment,there would be at least one mutation in the Lpx1 gene of the A, B and Dgenomes.

In another embodiment, the disclosure relates to a method for producinga wheat plant comprising crossing a first wheat plant that has at leastone non-transgenic mutation in a first Lpx1 gene with a second wheatplant that has at least one non-transgenic mutation in a second Lpx1gene; and selecting progeny wheat plants that have at least one mutationin at least two copies of an Lpx1 gene.

In another embodiment, the disclosure relates to a method for producinga wheat plant comprising crossing a first wheat plant that has at leastone non-transgenic mutation in a first and second Lpx1 gene with asecond wheat plant that has at least one non-transgenic mutation in athird Lpx1 gene; and selecting progeny wheat plants that have at leastone mutation in all three copies of an Lpx1 gene. In this embodiment,there would be at least one mutation in the Lpx1 gene of the A, B and Dgenomes.

VIII. Wheat Plant, Wheat Seed and Parts of Wheat Plant

In one embodiment, a wheat plant is produced according to the methodsdisclosed herein. In another embodiment, the wheat plant, wheat seed orparts of a wheat plant have one or more mutations in an Lpx1 gene. Inanother embodiment, the wheat plant, wheat seed or parts of a wheatplant have one or more mutations in Lpx1 genes.

In another embodiment, the disclosure relates to a wheat plant, wheatseed or parts of a wheat plant comprising one or more non-transgenicmutations in the Lpx1 gene. In another embodiment, the disclosurerelates to a wheat plant, wheat seed or parts of a wheat plantcomprising at least one non-transgenic mutation in the Lpx1 gene in eachof two genomes. In still another embodiment, the disclosure relates to awheat plant, wheat seed or parts of a wheat plant comprising at leastone non-transgenic mutation in the Lpx1 gene in each of three genomes.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theLpx1 gene in the A genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the Lpx1 gene of the Agenome.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theLpx1 gene in the B genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the Lpx1 gene of the Bgenome.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theLpx1 gene in the D genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the Lpx1 gene of the Dgenome.

In another embodiment, the wheat plant, wheat seed or parts of the wheatplant comprise a polynucleotide with one or more non-transgenicmutations listed in Table 1 and is 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical orsimilar to SEQ ID NO: 4.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprise a polynucleotide with one or more non-transgenicmutations listed in Table 1 that codes for a Lpx1 protein, wherein theLpx1 protein comprises one or more non-transgenic mutations and is 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 6.

In another embodiment, the wheat plant, wheat seed or parts of the wheatplant comprise a polynucleotide with one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical or similar to SEQ IDNO: 4.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprise a polynucleotide with one or more non-transgenicmutations that codes for a Lpx1 protein, wherein the Lpx1 proteincomprises one or more non-transgenic mutations and is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% identical or similar to SEQ ID NO: 6.

In another embodiment, the wheat plant, wheat seed or parts of the wheatplant comprise a polynucleotide with one or more non-transgenicmutations listed in Table 2 and is 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical orsimilar to SEQ ID NO: 1.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprise a polynucleotide with one or more non-transgenicmutations listed in Table 2 that codes for a Lpx1 protein, wherein theLpx1 protein comprises one or more non-transgenic mutations and is 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 3.

In another embodiment, the wheat plant, wheat seed or parts of the wheatplant comprise a polynucleotide with one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical or similar to SEQ IDNO: 2.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprise a polynucleotide with one or more non-transgenicmutations that codes for a Lpx1 protein, wherein the Lpx1 proteincomprises one or more non-transgenic mutations and is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% identical or similar to SEQ ID NO: 3.

In another embodiment, the wheat plant, wheat seed or parts of a wheatplant has one or more mutations in the Lpx1 gene including but notlimited to one or more mutations enumerated in Tables 1-3 andcorresponding mutations in the homoeologues. A wheat plant, wheat seedor parts of a wheat plant can be generated having 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 orgreater than 25 of the mutations disclosed herein including but notlimited to the mutations disclosed in Tables 1, 2 and 3, as well asmutations in the corresponding homoeologues.

In another embodiment, the wheat seed containing one or more mutationsdisclosed herein germinates at a rate comparable to wild type wheatseed. In still another embodiment, the wheat seed containing one or moremutations disclosed herein has physical characteristics, including butnot limited to size, weight, length, comparable to wild type wheat seed.

In still another embodiment, the wheat plants containing one or moremutations disclosed herein has fertility comparable wild type wheatplants.

IX. Grain, Flour and Starch

In another embodiment, the disclosure relates to a wheat grain, flour orstarch comprising one or more non-transgenic mutations in the Lpx1 gene.In another embodiment, the invention relates to wheat grain comprisingan embryo, wherein the embryo comprises one or more non-transgenicmutations in an Lpx1 gene.

In another embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in the Lpx1 genes including but notlimited to the mutations recited in Tables 1-3 and the correspondingmutations in homoeologues.

In still another embodiment, the disclosure relates to a wheat grain orflour comprising at least one non-transgenic mutation in the Lpx1 genein one, two or three genomes.

In still another embodiment, the invention relates to a wheat grain,flour or starch comprising one or more non-transgenic mutations in theLpx-D1 gene. In another embodiment, the non-transgenic mutations areidentical in both alleles of the Lpx-D1 gene of the D genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the Lpx-B1.1a gene inthe B genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the Lpx-B1.1a gene of the B genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the Lpx-B1.1b gene inthe B genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the Lpx-B1.1b gene of the B genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the Lpx-B1.1c gene inthe B genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the Lpx-B1.1c gene of the B genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the Lpx-B1.2 gene inthe B genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the Lpx-B1.2 gene of the B genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the Lpx-B1.3 gene inthe B genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the Lpx-B1.3 gene of the B genome.

In one embodiment, the disclosure relates to wheat grain, wheat flour orstarch comprising a polynucleotide of the Lpx-D1 promoter in the Dgenome with one or more non-transgenic mutations listed in Table 3 andcorresponding to SEQ ID NO: 15. In another embodiment, the wheat grainor wheat flour comprise a polynucleotide with one or more non-transgenicmutations listed in Table 3 and is 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical orsimilar to SEQ ID NO: 15.

In one embodiment, the disclosure relates to wheat grain, wheat flour orstarch comprising a polynucleotide of the Lpx-D1 gene in the D genomewith one or more non-transgenic mutations listed in Table 2 andcorresponding to SEQ ID NO: 1. In another embodiment, the wheat grain orwheat flour comprise a polynucleotide with one or more non-transgenicmutations listed in Table 2 and is 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical orsimilar to SEQ ID NO: 1.

In still another embodiment, wheat grain, wheat flour or starch comprisea polynucleotide with one or more non-transgenic mutations listed inTable 2 that codes for a Lpx1 protein, wherein the Lpx1 proteincomprises one or more non-transgenic mutations and is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% identical or similar to SEQ ID NO: 3.

In one embodiment, the disclosure relates to wheat grain, wheat flour orstarch comprising a polynucleotide of the Lpx-B1.2 gene in the B genomewith one or more non-transgenic mutations listed in Table 1 andcorresponding to SEQ ID NO: 4. In another embodiment, the wheat grain orwheat flour comprise a polynucleotide with one or more non-transgenicmutations listed in Table 1 and is 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical orsimilar to SEQ ID NO: 4.

In still another embodiment, wheat grain, wheat flour or starch comprisea polynucleotide with one or more non-transgenic mutations listed inTable 1 that codes for a Lpx-B1.2 protein, wherein the Lpx-B1.2 proteincomprises one or more non-transgenic mutations and is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% identical or similar to SEQ ID NO: 6.

In still another embodiment, the disclosure relates to wheat grain orflour comprising an endosperm and a reduced gene expression level,activity or expression level and activity of the Lpx1 gene as comparedto wild type wheat grain or flour.

In yet another embodiment, the disclosure relates to wheat grain orflour with one or more mutations in the Lpx1 gene exhibiting increasedshelf life as compared to wild type wheat grain or flour. In anotherembodiment, wheat grain or flour with one or more mutations in the Lpx1gene exhibits from 25-30%, 30-35%, 35-40%, 45-50%, 50-55%, 55-60%,60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, and greater than95% increased shelf life as compared to wild type grain or flour.

In yet another embodiment, the disclosure relates to wheat grain orflour with one or more mutations in the Lpx-D1 gene exhibiting increasedshelf life as compared to wild type wheat grain or flour. In anotherembodiment, wheat grain or flour with one or more mutations in theLpx-D1 gene exhibits from 25-30%, 30-35%, 35-40%, 45-50%, 50-55%,55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, andgreater than 95% increased shelf life as compared to wild type grain orflour.

In yet another embodiment, the disclosure relates to wheat grain orflour with one or more mutations in the Lpx-D1 and Lpx-B1.2 genesexhibiting increased shelf life as compared to wild type wheat grain orflour. In another embodiment, wheat grain or flour with one or moremutations in the Lpx-D1 and Lpx-B1.2 genes exhibits from 25-30%, 30-35%,35-40%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%,85-90%, 90-95%, and greater than 95% increased shelf life as compared towild type grain or flour.

X. Food Products

In one embodiment, the disclosure is directed to a flour or otherproduct produced from the grain or flour discussed above. In anotherembodiments, the flour, the coarse fraction or purified starch may be acomponent of a food product.

The food product includes but is not limited to a bagel, a biscuit, abread, a bun, a croissant, a dumpling, an English muffin, a muffin, apita bread, a quickbread, a refrigerated/frozen dough products, dough,baked beans, a burrito, chili, a taco, a tamale, a tortilla, a pot pie,a ready to eat cereal, a ready to eat meal, stuffing, a microwaveablemeal, a brownie, a cake, a cheesecake, a coffee cake, a cookie, adessert, a pastry, a sweet roll, a candy bar, a pie crust, pie filling,baby food, a baking mix, a batter, a breading, a gravy mix, a meatextender, a meat substitute, a seasoning mix, a soup mix, a gravy, aroux, a salad dressing, a soup, sour cream, a noodle, a pasta, ramennoodles, chow mein noodles, lo mein noodles, an ice cream inclusion, anice cream bar, an ice cream cone, an ice cream sandwich, a cracker, acrouton, a doughnut, an egg roll, an extruded snack, a fruit and grainbar, a microwaveable snack product, a nutritional bar, a pancake, apar-baked bakery product, a pretzel, a pudding, a granola-based product,a snack chip, a snack food, a snack mix, a waffle, a pizza crust, animalfood or pet food.

In one embodiment, the flour is a whole grain flour (ex.—anultrafine-milled whole grain flour, such as an ultrafine-milled wholegrain wheat flour). In one embodiment, the whole grain flour includes arefined flour constituent (ex.—refined wheat flour or refined flour) anda coarse fraction (ex.—an ultrafine-milled coarse fraction). Refinedwheat flour may be flour which is prepared, for example, by grinding andbolting (sifting) cleaned wheat. The Food and Drug Administration (FDA)requires flour to meet certain particle size standards in order to beincluded in the category of refined wheat flour. The particle size ofrefined wheat flour is described as flour in which not less than 98%passes through a cloth having openings not larger than those of wovenwire cloth designated “212 micrometers (U.S. Wire 70).”

In another embodiment, the coarse fraction includes at least one of:bran and germ. For instance, the germ is an embryonic plant found withinthe wheat kernel. The germ includes lipids, fiber, vitamins, protein,minerals and phytonutrients, such as flavonoids. The bran may includeseveral cell layers and has a significant amount of lipids, fiber,vitamins, protein, minerals and phytonutrients, such as flavonoids.

For example, the coarse fraction or whole grain flour or refined flourof the present invention may be used in various amounts to replacerefined or whole grain flour in baked goods, snack products, and foodproducts. The whole grain flour (i.e.—ultrafine-milled whole grainflour) may also be marketed directly to consumers for use in theirhomemade baked products. In an exemplary embodiment, a granulationprofile of the whole grain flour is such that 98% of particles by weightof the whole grain flour are less than 212 micrometers.

In another embodiment, the whole grain flour or coarse fraction orrefined flour may be a component of a nutritional supplement. Thenutritional supplement may be a product that is added to the dietcontaining one or more ingredients, typically including: vitamins,minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices,probiotics, extracts, prebiotics and fiber.

In a further embodiment, the nutritional supplement may include anyknown nutritional ingredients that will aid in the overall health of anindividual, examples include but are not limited to carotenoids,vitamins, minerals, other fiber components, fatty acids, antioxidants,amino acids, peptides, proteins, lutein, ribose, omega-3 fatty acids,and/or other nutritional ingredients. Because of the high nutritionalcontent of the endosperm of the present invention, there may be manyuses that confer numerous benefits to an individual, including, deliveryof fiber and other essential nutrients, increased digestive function andhealth, weight management, blood sugar management, heart health,diabetes risk reduction, potential arthritis risk reduction, and overallhealth and wellness for an individual.

In still another embodiments, the whole grain flour or coarse fractionor refined flour may be a component of a dietary supplement. The Code ofFederal Regulations defines a dietary supplement as a product that isintended to supplement the diet and contains one or more dietaryingredients including: vitamins, minerals, herbs, botanicals, aminoacids, and other substances or their constituents; is intended to betaken by mouth as a pill, capsule, tablet, or liquid; and is labeled onthe front panel as being a dietary supplement.

In yet another embodiment, the whole grain flour or coarse fraction orrefined flour may be a fiber supplement or a component thereof. Thefiber supplement may be delivered in, but is not limited to thefollowing forms: instant beverage mixes, ready-to-drink beverages,nutritional bars, wafers, cookies, crackers, gel shots, capsules, chews,chewable tablets, and pills. One embodiment delivers the fibersupplement in the form of a flavored shake or malt type beverage.

In another embodiment, the whole grain flour or coarse fraction orrefined flour may be included as a component of a digestive supplement.The whole grain flour or coarse fraction or refined flour may be acomponent of a digestive supplement alone or in combination with one ormore prebiotic compounds and/or probiotic organisms. Prebiotic compoundsare non-digestible food ingredients that may beneficially affect thehost by selectively stimulating the growth and/or the activity of alimited number of microorganisms in the colon. Examples of prebioticcompounds within the scope of the invention, may include, but are notlimited to: oligosaccharides and inulins.

Probiotics are microorganisms which, when administered in adequateamounts, confer a health benefit on the host. Probiotic organismsinclude, but are not limited to: Lactobacillus, Bifidobacteria,Escherichia, Clostridium, Lactococcus, Streptococcus, Enterococcus, andSaccharomyces.

In yet another embodiment, the whole grain flour or coarse fraction orrefined flour may be included as a component of a functional food. TheInstitute of Food Technologists defines functional foods as, foods andfood components that provide a health benefit beyond basic nutrition.This includes conventional foods, fortified, enriched, or enhancedfoods, and dietary supplements. The whole grain flour and coarsefraction or refined flour include numerous vitamins and minerals, havehigh oxygen radical absorption capacities, and are high in fiber, makingthem ideally suited for use in/as a functional food.

In another embodiment, the whole grain flour or coarse fraction orrefined flour may be used in medical foods. Medical food is defined as afood that is formulated to be consumed or administered entirely underthe supervision of a physician and which is intended for the specificdietary management of a disease or condition for which distinctivenutritional requirements, based on recognized scientific principles, areestablished by medical evaluation. The nutrient contents and antioxidantcapacities of the whole grain flour and coarse fraction or refined flourmake them ideal for use in medical foods.

In yet another embodiment, the whole grain flour or coarse fraction orrefined flour may also be used in pharmaceuticals. The whole grain flourand coarse fraction or refined flour are high in fiber and have a veryfine granulation making them suitable for use as a carrier inpharmaceuticals.

In still another embodiment, delivery of the whole grain flour or coarsefraction or refined flour as a nutritional supplement, dietarysupplement or digestive supplement is contemplated via deliverymechanisms where the whole grain flour or coarse fraction is the singleingredient or one of many nutritional ingredients. Examples of deliverymechanisms include but are not limited to: instant beverage mixes,ready-to-drink beverages, nutritional bars, wafers, cookies, crackers,gel shots, capsules, and chews.

In yet another embodiment, a milling process may be used to make amulti-wheat flour, or a multi-grain coarse fraction. In one embodiment,bran and germ from one type of wheat may be ground and blended withground endosperm or whole grain wheat flour of another type of wheat.Alternatively bran and germ of one type of grain may be ground andblended with ground endosperm or whole grain flour of another type ofgrain.

In still another embodiment, bran and germ from a first type of wheat orgrain may be blended with bran and germ from a second type of wheat orgrain to produce a multi-grain coarse fraction. It is contemplated thatthe invention encompasses mixing any combination of one or more of bran,germ, endosperm, and whole grain flour of one or more grains. Thismulti-grain, multi-wheat approach may be used to make custom flour andcapitalize on the qualities and nutritional contents of multiple typesof grains or wheats to make one flour.

The whole grain flour of the invention may be produced via a variety ofmilling processes. One exemplary process involves grinding grain in asingle stream without separating endosperm, bran, and germ of the graininto separate streams. Clean and tempered grain is conveyed to a firstpassage grinder, such as a hammermill, roller mill, pin mill, impactmill, disc mill, air attrition mill, gap mill, or the like.

After grinding, the grain is discharged and conveyed to a sifter. Anysifter known in the art for sifting a ground particle may be used.Material passing through the screen of the sifter is the whole grainflour of the invention and requires no further processing. Material thatremains on the screen is referred to as a second fraction. The secondfraction requires additional particle reduction. Thus, this secondfraction may be conveyed to a second passage grinder.

After grinding, the second fraction may be conveyed to a second sifter.Material passing through the screen of the second sifter is the wholegrain flour. The material that remains on the screen is referred to asthe fourth fraction and requires further processing to reduce theparticle size. The fourth fraction on the screen of the second sifter isconveyed back into either the first passage grinder or the secondpassage grinder for further processing via a feedback loop.

It is contemplated that the whole grain flour, coarse fraction, purifiedstarch and/or grain products of the invention may be produced by anumber of milling processes known in the art.

XI. Plant Breeding

In another embodiment, the disclosure is directed to methods for plantbreeding using wheat plants and plant parts with one or morenon-transgenic mutations in the Lpx1 genes.

One such embodiment is the method of crossing wheat variety with one ormore non-transgenic mutations in the Lpx1 genes with another variety ofwheat to form a first generation population of F1 plants. The populationof first generation F1 plants produced by this method is also anembodiment of the invention. This first generation population of F1plants will comprise an essentially complete set of the alleles of wheatvariety with one or more non-transgenic mutations in the Lpx1 genes. Oneof ordinary skill in the art can utilize either breeder books ormolecular methods to identify a particular F1 plant produced using wheatvariety with one or more non-transgenic mutations in the Lpx1 genes, andany such individual plant is also encompassed by this invention. Theseembodiments also cover use of transgenic or backcross conversions ofwheat varieties with one or more mutations in the Lpx1 genes to producefirst generation F1 plants.

In another embodiment, the invention relates to a method of developing aprogeny wheat plant. A method of developing a progeny wheat plantcomprises crossing a wheat variety with one or more non-transgenicmutations in the Lpx1 genes with a second wheat plant and performing abreeding method. A specific method for producing a line derived fromwheat variety with one or more non-transgenic mutations in the Lpx1genes is as follows.

One of ordinary skill in the art would cross wheat variety with one ormore non-transgenic mutations in the Lpx1 gene or genes with anothervariety of wheat, such as an elite variety. The F1 seed derived fromthis cross would be grown to form a homogeneous population. The F1 seedwould contain one set of the alleles from wheat variety with one or morenon-transgenic mutations in the Lpx1 gene and one set of the allelesfrom the other wheat variety.

The F1 genome would be made-up of 50% wheat variety with one or morenon-transgenic mutations in the Lpx1 gene and 50% of the other elitevariety. The F1 seed would be grown to form F2 seed. The F1 seed couldbe allowed to self, or bred with another wheat cultivar.

On average the F2 seed would have derived 50% of its alleles from wheatvariety with one or more non-transgenic mutations in the Lpx1 gene and50% from the other wheat variety, but various individual plants from thepopulation would have a much greater percentage of their alleles derivedfrom wheat variety with one or more non-transgenic mutations in the Lpx1gene (Wang J. and R. Bernardo, 2000, Crop Sci. 40:659-665 and Bernardo,R. and A. L. Kahler, 2001, Theor. Appl. Genet. 102:986-992).

The F2 seed would be grown and selection of plants would be made basedon visual observation and/or measurement of traits and/or markerassisted selection. The wheat variety with one or more non-transgenicmutations in the Lpx1 gene-derived progeny that exhibit one or more ofthe desired wheat variety with one or more non-transgenic mutations inthe Lpx1 gene-derived traits would be selected and each plant would beharvested separately. This F3 seed from each plant would be grown inindividual rows and allowed to self. Then selected rows or plants fromthe rows would be harvested and threshed individually. The selectionswould again be based on visual observation and/or measurements fordesirable traits of the plants, such as one or more of the desirablewheat variety with one or more non-transgenic mutations in the Lpx1gene-derived traits.

The process of growing and selection would be repeated any number oftimes until a homozygous wheat variety with one or more non-transgenicmutations in the Lpx1 gene-derived wheat plant is obtained. Thehomozygous wheat variety with one or more non-transgenic mutations inthe Lpx1 gene-derived wheat plant would contain desirable traits derivedfrom wheat variety with one or more non-transgenic mutations in the Lpxgenes, some of which may not have been expressed by the other originalwheat variety to which wheat variety with one or more non-transgenicmutations in the Lpx1 gene was crossed and some of which may have beenexpressed by both wheat varieties but now would be at a level equal toor greater than the level expressed in wheat variety with one or morenon-transgenic mutations in the Lpx1 gene or genes.

The breeding process, of crossing, selfing, and selection may berepeated to produce another population of wheat variety with one or morenon-transgenic mutations in the Lpx1 gene-derived wheat plants with, onaverage, 25% of their genes derived from wheat variety with one or morenon-transgenic mutations in the Lpx1 gene, but various individual plantsfrom the population would have a much greater percentage of theiralleles derived from wheat variety with one or more non-transgenicmutations in the Lpx1 gene or genes. Another embodiment of the inventionis a homozygous wheat variety with one or more non-transgenic mutationsin the Lpx gene-derived wheat plant that has received wheat variety withone or more non-transgenic mutations in the Lpx gene-derived traits.This breeding process can be repeated as many times as desired.

The following paragraphs further describe plants, compositions, andmethods disclosed herein.

1. A wheat plant comprising a mutation in the Lpx1 gene, wherein saidmutation contributes to products from said wheat plant having increasedshelf-life compared to products from a wild type wheat plant.

2. A wheat plant comprising a mutation in the Lpx1 gene, wherein saidmutation contributes to reduced hexanal production in products producedfrom grain from said plant as compared to products produced from grainfrom a wild type wheat plant.

3. The wheat plant of paragraphs 1 or 2, further comprising a mutationin at least two genomes.

4. The wheat plant of paragraphs 1 or 2, further comprising a reducedlevel of Lpx1 protein, relative to a wild-type wheat plant.

5. The wheat plant of paragraphs 1 or 2, further comprising reduced Lpx1enzyme activity relative to a wild-type wheat plant.

6. The wheat plant of paragraphs 1 or 2, wherein products from saidwheat plant have increased oxidative stability as compared to productsfrom a wild type wheat plant.

7. A wheat plant comprising a mutation in the Lpx1 gene in the D genome,wherein said mutation contributes to products from said wheat planthaving increased shelf-life compared to products from a wild type wheatplant.

8. A wheat plant comprising a mutation in the Lpx1 gene in the D genome,wherein said mutation contributes to reduced hexanal production inproducts from grain from said plant as compared to products producedfrom grain from a wild type wheat plant.

9. The wheat plant of paragraphs 7 or 8, wherein the Lpx1 gene isLpx-D1.

10. The wheat plant of paragraphs 7 or 8, further comprising a mutationin the Lpx1 gene of the B genome.

11. The wheat plant of paragraphs 7 or 8, further comprising a reducedlevel of Lpx1 protein, relative to a wild-type wheat plant.

12. The wheat plant of paragraphs 7 or 8, further comprising reducedLpx1 enzyme activity relative to a wild-type wheat plant.

13. The wheat plant of paragraphs 7 or 8, wherein products from saidwheat plant have increased oxidative stability as compared to productsfrom a wild type wheat plant.

14. A wheat plant comprising a mutation in the Lpx1 gene in the Bgenome, wherein said mutation contributes to products from said wheatplant having increased shelf-life compared to products from a wild typewheat plant.

15. A wheat plant comprising a mutation in the Lpx1 gene in the Bgenome, wherein said mutation contributes to reduced hexanal productionin products from grain from said plant as compared to products fromgrain from a wild type wheat plant.

16. The wheat plant of paragraphs 14 or 15, wherein the Lpx1 gene isLpx-B1.2.

17. A wheat plant of paragraphs 14 or 15 further comprising a mutationin the Lpx1 gene of the D genome.

18. The wheat plant of paragraphs 14 or 15, further comprising a reducedlevel of Lpx1 protein, relative to a wild-type wheat plant.

19. The wheat plant of paragraphs 14 or 15, further comprising reducedLpx1 enzyme activity relative to a wild-type wheat plant.

20. The wheat plant of paragraphs 14 or 15, wherein products from saidwheat plant have increased oxidative stability as compared to productsfrom a wild type wheat plant.

21. The wheat plant of any of the preceding paragraphs where the wheatplant is homozygous for the mutation.

22. The wheat plant of any of the preceding paragraphs, which isTriticum aestivum ssp. aestivum.

23. A wheat plant comprising two or more mutations in the Lpx gene,wherein the mutations in the Lpx gene are on at least two differentgenomes.

24. Whole grain flour from grain from the wheat plant of any of thepreceding paragraphs, wherein the production of decomposition productsof fatty acids is decreased in whole grain flour as compared to wholegrain flour made from wild type grain.

25. Whole grain flour from grain from the wheat plant of any of thepreceding paragraphs, wherein the production of decomposition productsof fatty acids is decreased in whole grain flour by at least 5%, or atleast 10%, or at least 15%, or at least 20%, or at least 25%, or atleast 30%, or at least 35%, or at least 40%, or at least 45%, or atleast 50%, or at least 55%, or at least 60%, or at least 65%, or atleast 70%, or at least 75%, or at least 80%, or at least 85%, or atleast 90%, or at least 95% as compared to whole grain flour made fromwild type grain.26. Whole grain flour from the wheat plant of any of the precedingparagraphs, wherein the production of hexanal, or trans-2-nonenal, ortrihydroxydecanoic acid or combinations thereof is decreased in wholegrain flour by at least 5%, or at least 10%, or at least 15%, or atleast 20%, or at least 25%, or at least 30%, or at least 35%, or atleast 40%, or at least 45%, or at least 50%, or at least 55%, or atleast 60%, or at least 65%, or at least 70%, or at least 75%, or atleast 80%, or at least 85%, or at least 90%, or at least 95% as comparedto whole grain flour made from wild type grain.27. Whole grain flour from the grain of the wheat plant of any of thepreceding paragraphs, wherein shelf life of whole grain flour isincreased by 1 month, 2 months, 3 months, 4 months, 5 months, 6 months,7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, 24 months, 25months, 26 months, 27 months, 28 months, 29 months, 30 months, orgreater than 30 months as compared to the shelf life of whole grainflouor made from wild-type grain.28. Whole grain flour from grain comprising a mutation in an Lpx1 gene,wherein said mutation contributes to reduced hexanal production in wholegrain flour as compared to whole grain flour from a wild type wheatplant.29. Whole grain flour comprising a mutation in an Lpx1 gene, whereinsaid mutation contributes to increased shelf-life in whole grain flouras compared to whole grain flour from a wild type wheat plant.30. Whole grain flour comprising a mutation in an Lpx1 gene, whereinsaid mutation contributes to increased shelf-life in whole grain flourstored at a higher temperature as compared to whole grain flour from awild type wheat plant.31. Whole grain flour from the wheat plant of any of the precedingparagraphs, wherein shelf life of whole grain flour made from wheatgrain is improved as determined by sensory characteristics includingcolor, flavor, texture, aroma, performance or overall preference of thefinished product.32. The wheat plant of any of the preceding claims, wherein the mutationis recited in any one of Tables 1-10.33. Wheat grain from a wheat plant of any of the preceding paragraphs.34. Flour comprising wheat grain of any of the preceding paragraphs.35. A food product comprising a component of a wheat plant of any of thepreceding paragraphs.36. A wheat seed, plant part or progeny thereof from a wheat plant ofany of the preceding paragraphs.37. A wheat plant substantially as shown and described herein.38. Grain substantially as shown and described herein.39. Wheat seed, plant part or progeny thereof from a wheat plantsubstantially as shown and described herein.40. A wheat plant comprising one or more mutations in the Lpx1 gene inone or both of the B and D genomes, wherein milled grain from said wheatplant has a property selected from the group consisting of: (a)increased shelf-life; (b) increased oxidative stability; (c) decreasedproduction of Lpx1 protein; (d) decreased activity of the Lpx1 protein;(e) decreased hexanal production; (f) decreased pinellic acidproduction; (g) decreased decomposition products from fatty acids; or(h) improved sensory characteristics as compared to milled grain from awild type wheat plant.41. A wheat plant comprising one or more mutations in the Lpx1 gene inone or both of the B and D genomes, wherein products produced from grainfrom said wheat plant has a property selected from the group consistingof: (a) increased shelf-life; (b) increased oxidative stability; (c)decreased production of Lpx1 protein; (d) decreased activity of the Lpx1protein; (e) decreased hexanal production; (0 decreased pinellic acidproduction; (g) decreased decomposition products from fatty acids; or(h) improved sensory characteristics as compared to products producedfrom grain of a wild type wheat plant.42. A nucleic acid comprising a coding sequence that encodes apolypeptide having at least one mutation recited in Table 2 of SEQ IDNO:3.43. A nucleic acid comprising a coding sequence having at least onemutation in Table 2 of SEQ ID NO. 1 or SEQ ID NO. 2.44. A nucleic acid comprising a coding sequence that encodes apolypeptide having at least one mutation recited in Table 1 of SEQ IDNO:6.45. A nucleic acid comprising a coding sequence having at least onemutation in Table 1 of SEQ ID NO. 4 or SEQ ID NO. 5.

Example 1

This example describes the identification of novel alleles of Lpx1.

Mutagenesis

In accordance with one exemplary embodiment of the present invention,wheat seeds of the hexaploid cultivar (Triticum aestivum) Express werevacuum infiltrated in H₂O (approximately 1,000 seeds/100 ml H₂O forapproximately 4 minutes). The seeds were then placed on a shaker (45rpm) in a fume hood at room temperature. The mutagen ethylmethanesulfonate (EMS) was added to the imbibing seeds to finalconcentrations ranging from about 0.75% to about 1.2% (v/v). Followingan 18-hour incubation period, the EMS solution was replaced 4 times withfresh H₂O. The seeds were then rinsed under running water for about 4-8hours. Finally, the mutagenized seeds were planted (96/tray) in pottingsoil and allowed to germinate indoors. Plants that were four to sixweeks old were transferred to the field to grow to fully mature M1plants. The mature M1 plants were allowed to self-pollinate and thenseeds from the M1 plant were collected and planted to produce M2 plants.

A. DNA Preparation

DNA from the M2 plants produced in accordance with the above descriptionwas extracted and prepared in order to identify which M2 plants carrieda mutation at one or more of their Lpx1 loci. The M2 plant DNA wasprepared using methods and reagents based on the Qiagen® (Valencia,Calif.) DNeasy® 96 Plant Kit. Approximately 50 mg of frozen plant samplewas placed in each sample tube with a stainless steel bead, frozen inliquid nitrogen and ground 2 times for 45 seconds each at 21.5 Hz usingthe Retsch® Mixer Mill MM 300. Next, 300 μl of solution AP1 [Buffer AP1,solution DX and RNAse (100 mg/ml)] at 80° C. was added to each sample.The tubes were sealed and shaken for 15 seconds, then brieflycentrifuged at 5,200×g. Following the addition of 100 μl Buffer P3, thetubes were shaken for 15 seconds. The samples were placed in a freezerat −20° C. for at least 20 min. The samples were then centrifuged for 20minutes at 5,200×g. A filter plate was placed on the vacuum unit ofTecan Evo liquid handling robot and 400 μl of Buffer AW1 was added toeach well. Following the addition of a 300 μl aliquot of supernatant toeach well, vacuum was applied until dryness. Next, 650 μl of Buffer AW2was added to each well of the filter plate. The filter plate was placedon a square well block and centrifuged for 20 minutes at 5,200×g. Thefilter plate was then placed on a new set of sample tubes and 90 μl ofBuffer AE was applied to the filter. It was incubated at roomtemperature for 1 minute and then spun for 2 minutes at 5,200×g. Thisstep was repeated with an additional 90 μl Buffer AE. The filter platewas removed and the tubes containing the pooled filtrates were capped.The individual samples were then normalized to a DNA concentration of 5to 10 ng/μl for TILLING, or left un-normalized for genotypingapplications.

B. Tilling

The M2 wheat DNA was pooled into groups of two individual plants. TheDNA concentration for each individual within the pool was approximately2 ng/μl with a final concentration of 4 ng/μl for the entire pool. Then,5 μl of the pooled DNA samples (or 20 ng wheat DNA) was arrayed onmicrotiter plates and subjected to gene-specific PCR.

PCR amplification was performed in 15 μl volumes containing 20 ng pooledDNA, 0.75×ExTaq buffer (Clonetech, Mountain View, Calif.), 1.1 mMadditional MgCl₂, 0.3 mM dNTPs, 0.3 μM primers, 0.009 U Ex-Taq DNApolymerase (Clonetech, Mountain View, Calif.), 0.02 units DyNAzyme IIDNA Polymerase (Thermo Scientific), and if necessary 0.33M Polymer-AidePCR Enhancer (Sigma-Aldrich®). PCR amplification was performed using anMJ Research® thermal cycler as follows: 95° C. for 2 minutes; 8 cyclesof “touchdown PCR” (94° C. for 20 second, followed by annealing stepstarting at 70-68° C. for 30 seconds and decreasing 1° C. per cycle,then a temperature ramp of 0.5° C. per second to 72° C. followed by 72°C. for 1 minute); 25-45 cycles of 94° C. for 20 seconds, 63 or 65° C.for 30 seconds, ramp 0.5° C./sec to 72° C., 72° C. for 1-2 minutes; 72°C. for 8 minutes; 98° C. for 8 minutes; 80° C. for 20 seconds; 60 cyclesof 80° C. for 7 seconds −0.3 degrees/cycle.

PCR products (2-4 μl) were digested in 96-well plates. 3 μl of asolution containing 6 mM HEPES[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (pH 7.0), 6 mMMgCl₂, 6 mM NaCl, 0.012× Triton® X-100, 0.03 mg/ml of bovine serumalbumin, 0.5× T-Digest Buffer [Advanced Analytical Technologies, Inc(AATI), Ames, Iowa], 0.912 U each of Surveyor® Endonuclease and Enhancer(Transgenomic®, Inc.), and 0.5×dsDNA Cleavage Enzyme (AATI, Ames, Iowa)was added to the PCR product. Digestion reactions were incubated at 45°C. for 45 minutes. The specific activity of the Surveyor enzyme was 800units/μl, where a unit was defined by the manufacturer as the amount ofenzyme required to produce 1 ng of acid-soluble material from sheared,heat denatured calf thymus DNA at pH 8.5 in one minute at 37° C.Reactions were stopped by addition of 20 μl of Dilution Buffer E (AATI,Ames, Iowa) or 1×TE. The reactions were stored in the freezer until theywere run on the Fragment Analyzer™ (AATI, Ames, Iowa) CapillaryElectrophoresis System. Samples were run on the Fragment Analyzer™utilizing the DNF-920-K1000T Mutation Discovery Kit (AATI, Ames, Iowa)according to the manufacturer's protocol.

After electrophoresis, the assays were analyzed using PROSize® 2.0Software (AATI, Ames, Iowa). The gel image showed sequence-specificpattern of background bands common to all 96 lanes. Rare events, such asmutations, create new bands that stand out above the background pattern.Plants with bands indicative of mutations of interest were evaluated byTILLING individual members of a pool mixed with wild type DNA and thensequencing individual PCR products.

Example 2: Genotyping and Plant Breeding of Lpx1 Lines

Plants carrying mutations confirmed by sequencing were grown up asdescribed above (e.g., the M2 plant could be backcrossed or outcrossedmultiple times in order to eliminate background mutations andself-pollinated in order to create a plant that was homozygous for themutation) or crossed to another plant containing Lpx1 mutations in adifferent homoeolog. At each generation, the novel alleles werevalidated in the plant materials by extracting DNA, and genotyped bysequencing or by use of allele specific KASP (Kompetitive AlleleSpecific PCR) molecular markers (LGC Genomics, Beverly, Mass.) developedspecifically for alleles of interest.

KASP genotyping was performed on DNA extracted from young leaves asdescribe in Example 1. Each reaction consisted of 5 μl master mix (KASPHigh-Rox Universal 2× Master Mix, LGC Genomics) 0.14 μl KASP Assay Mix,and 40-60 ng DNA in a total reaction volume of 10.14 μl. The reactionmixture was then PCR amplified in a 96-well format using the followingthermal cycling conditions: 94° C. for 15 minutes, then 10 cycles at 92°C. for 20 seconds followed by 61° C. for 60 seconds dropping 0.6° C. percycle until reaching 55° C., then 35-40 cycles of 94° C. for 20 secondsfollowed by 55° C. for 60 seconds, and finally held at 8° C. untilmeasurement. The subsequent reaction was evaluated at room temperaturewith a 7900 HT Fast Real-Time PCR system using controls of knowngenotypes (Applied Biosystems, Inc, Foster City, Ca, USA).

Example 3: Lipoxygenase Activity

Lipoxygenase activity in mature whole grains was measured by either oftwo methods: (a) conjugated diene formation or (b) colorimetric assay.

A. Method 1: Lipoxygenase Enzyme Activity by Conjugated Diene Formation

For conjugated diene analysis, total lipoxygenase activity in wholegrain wheat flour was measured spectrophotometrically as described bySurrey, Plant Physiology 39: 65-70, (1964). The assay determines theformation of linoleate hydroperoxide containing conjugated dienes at 25°C. measured at 234 nm using a NanoDrop 2000c Spectrophotometer (ThermoScientific, Waltham, Mass., USA). Whole grain flour was milled frommature seeds for 20 sec at 22 1/s vibration frequency using a MixerMill300 (Retsch GmbH, Haan, Germany). 200 mg of whole grain flour wassuspended in 1.5 mL of 50 mM sodium phosphate buffer (pH 6.6). Thesuspension was incubated in an ice-water bath for 1 hour, vortexed for 1min at 15 min intervals, and centrifuged twice at 16,100×g at 4° C. fora total of 20 min. After centrifugation, the supernatant was collectedas the crude enzyme solution, stored in an ice-water bath, and usedwithin 12 hours. Protein concentration was determined according to theBradford method (Bio-Rad, Hercules, Calif., USA) using bovine serumalbumin as the standard according to the manufacturer's instructions.Linoleate substrate solution consisted of 50 mM sodium phosphate bufferpH 6.6, 2 mM linoleic acid >99% (Sigma Chemical, St Louis, Mo., USA),and 0.2% Tween20 (Bio-Rad, Hercules, Calif., USA), and was stored in anair tight container containing 5-7 oxygen scavenging packets (Oxyrase,Mansfield, Ohio, USA) to minimize auto-oxidation of substrate. Linoleatehydroperoxidation reaction was started by the addition of 0.1 mg ofcrude enzyme solution into 2 mL of linoleate substrate solution at 25°C. The reaction was monitored from 0 to at least 3 minutes. Results weresubjected to statistical analysis by ANOVA followed by t-tests usingInStat Software (GraphPad, La Jolla, Calif., USA). One unit ofLipoxygenase activity was defined as the increase in absorbance at 234nm of 0.001 per mg of protein per minute. Negative controls of thesamples were prepared by inactivating the crude enzymes by heattreatment at 100° C. for 20 min.

B. Method 2: Lipoxygenase Activity by Colorimetric Assay

Total lipoxygenase activity in whole grain wheat flour was also measuredusing a modified colorimetric assay using the coupled reaction of3-(di-methylamino)benzoic acid (DMAB) and 3-methyl-2-benzothiazolinone(MBTH) as described by Anton and Barret, Journal of Agriculture FoodChemistry 49: 32-37, (2001). The assay determines the product formationof linoleate hydroperoxides with MBTH and DMAB in the presence ofhemoglobin. Whole grain flour was milled from mature seeds for 20seconds at 22 1/s vibration frequency using a MixerMill 300 (RetschGmbH, Haan, Germany). 200 mg of whole grain flour was suspended in 1.5mL of 50 mM sodium phosphate buffer (pH 6.6). The suspension wasincubated in an ice-water bath for 1 hour, vortexed for 1 min at 15minute intervals, and centrifuged twice at 16,100×g at 4° C. for a totalof 20 minutes. After centrifugation, the supernatant was collected asthe crude enzyme solution, stored in an ice-water bath, and used within12 hours. Protein concentration was determined according to the Bradfordmethod (Bio-Rad, Hercules, Calif., USA) using bovine serum albumin asthe standard according to the manufacturer's instructions.DMAB-linoleate substrate solution consisted of 20 mM DMAB and 100 μMsodium phosphate (pH 6.6) with 25 mM linoleic acid >99% and 0.1%Tween20. MBTH-hemoglobin solution consisted of 10 mM MBTH and 0.1 mg/mLbovine hemoglobin (Sigma Chemical, St Louis, Mo., USA).

The linoleate hyroperoxidation/DMAB-MBTH coupling reaction was startedby adding 0.5 ml of the DMAB-linoleate substrate solution to 0.1 mgcrude enzyme and incubated at 25° C. for 20 minutes. After 20 minutes,0.5 ml of the MBTH-hemoglobin solution was then added and incubated foran additional 10 minutes. The reaction was terminated by adding 0.5 mlof 1% (w/v) sodium dodecyl sulfate (SDS). Color formation at anabsorbance value of 595 nm was measured using a NanoDrop 2000cSpectrophotometer (Thermo Scientific, Waltham, Mass., USA). Results weresubjected to statistical analysis using InStat Software (GraphPad, LaJolla, Calif., USA). One unit of lipoxygenase activity was defined asthe absorbance value at 595 nm per mg of protein times 100. Negativecontrols of the samples were prepared by inactivating the crude enzymesby heat treatment at 100° C. for 20 min.

Grains from homozygous wheat plants with mutations in either Lpx-D1 orLpxB1.2 of the D or B genome or both genomes were analyzed forlipoxygenase activity. In addition, selected plants identified withsevere mutations in Lpx1 of the B or D genome (Tables 1 and 2) werecrossed with other plants that contained severe mutations in Lpx1 inother genomes. Grains from homozygous plants resulting from thesecrosses having novel mutant alleles in both genomes were also analyzedfor lipoxygenase activity. Sibling plants from these crosses withwild-type alleles for the Lpx1 mutations were used as controls when theywere available. Mutant alleles analyzed for lipoxygenase activityincluded missense mutations that were predicted to have a deleteriouseffect on protein function by their SIFT and PSSM scores, as well asthose mutations that resulted in the introduction of a stop codon(truncation mutation) or a mutation at a splice junction. Table 5 showsexamples of mutant lines analyzed for lipoxygenase activity.

TABLE 5 Representative cultivars with mutations in Lpx-D1 and/orLpx-B1.2 Nucleotide A.A. Line Gene Mutation Mutation 1 Lpx-D1 G626A W81*2 Lpx-D1 G685A W101* 3 Lpx-D1 C1471T P170S 4 Lpx-D1 G1646A R228H 5Lpx-D1 C1531T Q190* 6 Lpx-D1 C1633T P224S 7 Lpx-D1 G2386A SpliceJunction 8 Lpx-D1 C2553T P469L 9 Lpx-D1 G2629A W494* 10 Lpx-D1 G2828AW517* 11 Lpx-D1 G2875A R533Q 12 Lpx-D1 C2895T P540S 13 Lpx-D1 C2919TH548Y 14 Lpx-D1 G3049 W591* 15 Lpx-D1 G3181A W635* 16 Lpx-D1 G3272AW665* 17 Lpx-D1 G3275A W666* 18 Lpx-D1 G3364A C696Y 19 Lpx-D1 G3379AW701* 20 Lpx-D1 G3644A W789* 21 Lpx-B1.2 G2982A W510* 22 Lpx-D1 G2629AW494* Lpx-B1.2 G2982A W510* 23 Lpx-D1 C2528T P461S Lpx-B1.2 G2933A W494*

With regard to Tables 5-7, genomic nucleic acid designations of themutations in Lpx1 of the D genome named Lpx-D1 correspond to theposition in the reference sequence SEQ ID NO: 1. Amino acid designationsof the Lpx1 polypeptide of the D genome named Lpx-D1 correspond to theamino acid position of reference sequence SEQ ID NO: 3. Genomic nucleicacid designations of the mutations in one of two of Lpx1 genes of the Bgenome named Lpx-B1.2 correspond to the position in the referencesequence SEQ ID NO: 4. Amino acid designations of the mutations in oneof two of Lpx1 genes of the B genome named Lpx-B1.2 correspond to theamino acid position of reference sequence SEQ ID NO: 6. “Wt” indicatesmaterial that is homozygous for the parental wild-type allele and“homozygous” refers to material that is homozygous for the mutant alleleindicated.

TABLE 6 Lipoxygenase Activity of Novel Alleles of Lpx1 in Wheat VarietyExpress. Lipoxygenase Activity Lipoxygenase Colorimetric Activity AssayConjugated Nucleotide A.A. Zygosity of (Units/mg Diene Assay Line GeneMutation Mutation Mutation protein) (Units/min/mg) Parent Lpx-D1 + WtNone Wt 1915 +/− 29 1231 +/− 62  Express Lpx-B1.2 1 Lpx-D1 G626A W81*Homozygous  709 +/− 47 2 Lpx-D1 G685A W101* Homozygous  593 +/− 63 3Lpx-D1 C1471T P170S Homozygous 1912 +/− 25 4 Lpx-D1 C1531T Q190*Homozygous 1039 +/− 40 5 Lpx-D1 G1646A R228H Homozygous 1879 +/− 29 6Lpx-D1 C1633T P224S Homozygous 1873 +/− 18 7 Lpx-D1 G2386A SpliceJunction Homozygous  358 +/− 21 8 Lpx-D1 C2553T P469L Homozygous 1883+/− 18 9 Lpx-D1 G2629A W494* Homozygous 502 +/− 7 33 +/− 26 10 Lpx-D1G2828A W517* Homozygous 401 +/− 2 80 +/− 32 11 Lpx-D1 G2875A R533QHomozygous 1690 +/− 66 476 +/− 56  12 Lpx-D1 C2895T P540S Homozygous1418 +/− 44 427 +/− 13  13 Lpx-D1 C2919T H548Y Homozygous 1763 +/− 3  14Lpx-D1 G3049A W591* Homozygous  707 +/− 90 15 Lpx-D1 G3181A W635*Homozygous  866 +/− 68 16 Lpx-D1 G3272A W665* Homozygous  681 +/− 56 17Lpx-D1 G3275A W666* Homozygous 1441 +/− 41 18 Lpx-D1 G3364A C696YHomozygous 1304 +/− 61 19 Lpx-D1 G3379A W701* Homozygous  681 +/− 160 20Lpx-D1 G1646A + G3644A R228H + W789* Both  799 +/− 10 Homozygous 21Lpx-B1.2 G2982A W510* Homozygous 1860 +/− 40 798 +/− 105 22 LpxD1 +G2629A + G2982A W494* + W510* Homozygous 387 +/− 2 Lpx-B1.2 G2629G +G2982G W494W + W510W Wt Sibling 1914 +/− 2  23 Lpx-D1 + C2528T + G2933AP461S + W494* Both 1912 +/− 2  853 +/− 109 Lpx-B1.2 Homozygous

Table 6 demonstrates the range of lipoxygenase activity of various lineswith novel alleles in the Lpx-D1 gene, the Lpx-B1.2 gene or combinationsof the two.

TABLE 7 Lipoxygenase Activity of Various Varieties of Wheat. Activity isexpressed in Units/mg protein using the colorimetric assay (+/− standarderror). Lipoxygenase Wheat Variety Type D Genome B Genome B Genome AGenome Activity California HRS Lpx-D1 Lpx-B1.1c Lpx-B1.2 Lpx-A1 1915 +/−29 Mexico HWS Lpx-D1 Lpx-B1.1c Lpx-B1.2 Lpx-A1 2024 +/− 38 Mexico HWSLpx-D1 Lpx-B1.1c Lpx-B1.2 Lpx-A1 1863 +/− 61 Colorado HWW Lpx-D1Lpx-B1.1c Lpx-B1.2 Lpx-A1 2032 +/− 32 Colorado HWW Lpx-D1 Lpx-B1.1cLpx-B1.2 Lpx-A1 2020 +/− 18 Midwest HRS Lpx-D1 Lpx-B1.1a or b Lpx-B1.2Lpx-A1 1935 +/− 79 Midwest HRS Lpx-D1 Lpx-B1.1a or b Lpx-B1.2 Lpx-A11955 +/− 15 Pacific Northwest HRS Lpx-D1 Lpx-B1.1c Lpx-B1.2 Lpx-A1 1958+/− 50 Pacific Northwest SWS Lpx-D1 Lpx-B1.1a or b Lpx-B1.3 Lpx-A1 1960+/− 15 Australia SWS Lpx-D1 Lpx-B1.1c Lpx-B1.2 Lpx-A1 2051 +/− 23Australia SWS Lpx-D1 Lpx-B1.1c Lpx-B1.2 Lpx-A1 1973 +/− 17 Key: HRS,hard red spring, HWW, hard white winter, SWS, soft white spring, HWS,hard white spring

Table 7 demonstrates the range of lipoxygenase activity achieved invarious combinations of novel alleles in the Lpx-D1 gene, the Lpx-B1.2gene and combinations of the two. Table 7 further demonstrates thatdespite the range of different allele combinations available in theLpx-B genes in multiple varieties of bread wheat, all the varietiestested still have a high level of lipoxygenase activity in maturegrains. Evaluation of numerous additional varieties from multipleregions around the world has shown the same result.

Example 4 Improved Shelf Life by Sensory Characteristics of Lpx1 NovelAlleles

Shelf life can be determined by sensory characteristics of the flour andproducts made from it including color, flavor, texture, aroma,appearance, performance or overall preference of the finished product.In one embodiment, trained panelists can be used to assess differencesbetween materials.

For example, Lpx1 flour can be stored for various lengths of time, atvarious temperatures and/or humidities and compared to the wild-typesibling flour and/or parental flour by the panelists for preference inaroma, color, flavor, appearance and texture among other attributes. Theflour can also be made into products, such as bread, and the crumb andcrust compared for in aroma, color, flavor, appearance or texture amongother attributes. Bread or other products can also be stored for variouslengths of time, at various temperatures and/or humidities and comparedto the wild-type sibling flour and/or parental flour by the panelistsfor preference in in aroma, color, flavor, appearance or texture amongother attributes.

Other methods can also be employed to assess sensory characteristics.For example, texture can be measured by a texture analyzer. Color can bemeasured by a Minolta chroma meter test. Compounds contributing to aromaor taste can be analyzed by liquid or gas chromatography and massspectrometry.

Example 5: Improved Shelf-Life of Lpx1 Novel Alleles

Lipid oxidation by lipoxygenase produces hydroperoxides that aresubstrates for further decomposition into multiple compounds includingaldehydes such as hexanal. Hexanal levels produced in a sample can beused as a measure of oxidative rancidity (Fritz and Gale, Hexanal as ameasure of rancidity in low fat foods, JAOCS 54:225 (1977)). In order totest shelf-life of whole grain flour derived from grain of novellipoxygenase mutant alleles, whole grain samples were milled and storedfor various lengths of time up to 20 weeks at 37° C. and analyzed forhexanal levels as described below. Longer incubation times and a rangeof additional temperature and humidity conditions can also be employedfor testing shelf life.

Methods: Hexanal Analysis

Whole grain flour was milled from mature seeds for 20 seconds at 22 1/svibration frequency using a MixerMill 300 (Retsch GmbH, Haan, Germany)and stored in closed polyethylene bags. Accelerated aging of flour wasconducted in a Percival E30BC8 (Percival Scientific Inc, Perry, Iowa,USA) with the temperature set at 37° C. 10 g samples of flour werestored for 1-16 weeks at 37° C. Hexanal levels were analyzed byMedallion Labs (Minneapolis, Minn., USA) using a method based on gaschromatography. Units were reported in parts per million (ppm) with alower detection limit of <0.3 ppm and an upper limit of detection of 50ppm.

An accelerated aging time-course of whole grain flour incubated at 37°C. for up to 20 weeks was evaluated for hexanal production as anindicator of rancidity. Flour from the non-mutagenized parental variety,Express, was tested at 1, 6, 8, 10, 16 and 20 weeks after incubation at37° C. and compared to hexanal levels in freshly milled flour samples.

As shown in FIG. 1, hexanal levels were below the limit of detection of<0.3 ppm in freshly ground and 1 week old samples. But starting at the 6week time point and continuing to 20 weeks, hexanal levels increasedfrom 0.6 ppm up to 1 ppm demonstrating the progression of oxidativerancidity. For cereals, a 16 week incubation time at 37° C. is estimatedto be equivalent to approximately 1 year of storage at room temperature(Sewald and DeVries, Food product shelf life, Medallion LaboratoriesAnalytical Progress (2012)http://www.medlabs.com/Downloads/food_product_shelf_life_web.pdf).

Wheat line 9 (Table 6) homozygous for the Lpx-D1(W494*) allele and wheatline 22 (Table 6) homozygous for both the Lpx-D1(W494*) andLpx-B1.2(W510*) alleles were tested for hexanal production in wholegrain flour stored at 37° C. for up to 16 weeks. In addition, siblinglines that were homozygous wild-type for these alleles and were grown atthe same time were used as controls (wild-type siblings). Grains werebulked from 2-6 plants of the same genotype to provide enough materialfor analysis over the entire time-course.

As shown in FIG. 2, hexanal levels increased at 6, 8 and 16 weeks ofincubation at 37° C. in wild-type siblings. This result was similar tohexanal production in the parental material (FIG. 1). In contrast,hexanal levels remained below the limit of detection of <0.3 ppm in thewhole grain flour samples from lines homozygous for the Lpx-D1(W494*)mutant allele and in lines homozygous for both theLpx-D1(W494*)+LpxB1.2(W510*) mutant alleles for up to 16 weeks at 37° C.(asterisks in FIG. 2). The disclosure herein demonstrates that novelalleles in Lpx-D1 with reduced lipoxygenase activity singly and incombination with novel alleles in Lpx-B1.2 significantly improveshelf-life of whole grain flour by reducing the accumulation ofbreakdown products of fatty acids such as hexanal. Additional alleles ofLpx1 can also be used to improve shelf life.

Example 6 Altered Lipoxygenase Activity of Wheat Lines with Lpx-D1Promoter Alleles

Table 8 provides additional examples of mutations created and identifiedin Lpx-D1 promoter in the D genome of wheat plants, variety Express.Nucleotide changes are identified according to their positions in SEQ IDNO: 15. Zygosity refers to whether the mutation is heterozygous (Het) orhomozygous (Hom) in the M2 plant.

TABLE 8 Additional representative mutations in the Lpx-D1 promoter inthe D genome. Nucleotide Mutation Primer (SEQ ID Zygosity of SEQ IDs NO:15) Description Mutation 16, 17 G1269A 2176F08 Het 16, 17 C1276T 2168C09Het 16, 17 G1474A 2163B03 Het 16, 17 C1779T 2166F07 Hom 16, 17 C1834T1686B02 Hom

Table 9 demonstrates the range of lipoxygenase activity achieved inwheat lines with novel alleles in the Lpx-D1 promoter region in the Dgenome. Nucleotide changes of the mutations correspond to the positionin the reference sequence SEQ ID NO: 15. “Wt” indicates material that ishomozygous for the parental wild-type allele and “homozygous” refers tomaterial that is homozygous for the mutant allele indicated. In Table 9,Line 5 Lpx-D1 promoter allele (G1538A) has reduced lipoxygenase activitycompared to the parent variety and other Lpx-D1 promoter alleles.

TABLE 9 Lipoxygenase activity of novel alleles of Lpx1-D1 promoter inthe D genome of wheat Lipoxygenase Activity Nucleotide ColorimetricMutation Assay (SEQ ID Zygosity of (Units/mg Line Gene NO: 15) Mutationprotein) Parent Lpx-D1 Promoter Wt Wt 2045 +/− 34 Express 1 Lpx-D1Promoter G1269A Homozygous 1956 +/− 36 G1269G Wt Sibling  1922 +/− 8.5 2Lpx-D1 Promoter C1276T Homozygous 1941 +/− 17 C1276C Wt Sibling 1935 +/−18 3 Lpx-D1 Promoter C1339T Homozygous 1947 +/− 22 4 Lpx-D1 PromoterG1474A Homozygous 2034 +/− 21 G1474G Wt Sibling 2092 +/− 40 5 Lpx-D1Promoter G1538A Homozygous  736 +/− 47 6 Lpx-D1 Promoter C1779THomozygous 1996 +/− 17 7 Lpx-D1 Promoter C1834T Homozygous 1957 +/− 36 8Lpx-D1 Promoter G1966A Homozygous 1983 +/− 9  G1966G Wt Sibling 1977 +/−19

Example 7 Improved Shelf-Life of Multiple Lpx-D1 Novel Alleles in 8 WeekAccelerated Aging Studies

Hexanal is a degradation product of fatty acids that can be used as ameasure of oxidative rancidity. Grains from plants with differentalleles in Lpx-D1 were tested for hexanal levels after milling andaccelerated aging at 37° C. for 8 weeks as described in Example 5. Theseincluded single allele wheat lines from Table 5 including Line 1homozygous for the Lpx-D1(W81*) allele, Line 6 homozygous for theLpx-D1(P2245) allele, Line 8 homozygous for the Lpx-D1(P469L) allele,Line 9 homozygous for the Lpx-D1(W494*) allele, Line 7 homozygous forthe Lpx-D1(Splice Junction) allele, Line 11 homozygous for theLpx-D1(R533Q) allele, Line 12 homozygous for the Lpx-D1 (P540S), Line 15homozygous for the Lpx-D1(W635*) allele, Line 17 homozygous for theLpx-D1(W666*) allele, Line 18 homozygous for the Lpx-D1(C696Y) alleleand Line 20 homozygous for the Lpx-D1(W789*) allele.

In addition, grain from the Parent Line Express (Parent) and siblinglines of wheat Lines 8, 9 and 15 that had wild-type alleles were grownat the same time, milled and used as controls (Wild-type Siblings).Hexanal levels were measured at Medallion Labs as described in Example5. As shown in FIG. 3, the Parent line and Wild-type Siblings lines hadelevated levels of hexanal after 8 weeks accelerated aging of wholegrain flour, whereas all single homozygous Lpx-D1 lines had reducedhexanal levels. Lines 1, 8, 9, 7, 11, 15, 17, 18 and 20 had hexanallevels below the level of detection (<0.3 ppm), whereas Lines 6 and 12had slightly elevated hexanal levels of 0.311 and 0.351 ppm.

In order to further test reduced rancidity and increased shelf-life ofwhole grain flour derived from grain of novel lipoxygenase mutantalleles, wheat Line 9 (Table 5) homozygous for the Lpx-D1(W494*) alleleand wheat Line 22 (Table 5) homozygous for both the Lpx-D1(W494*) andLpx-B1.2(W510*) alleles were milled as described in Example 5. Inaddition, sibling lines of wheat Lines 9 or 22 that had homozygouswild-type alleles were milled and used as controls (wild-type siblings).Triplicate biological repeats of whole grain flour were subjected toaccelerated aging for 8 weeks and analyzed for hexanal levels asdescribed in Example 5. As shown in FIG. 4, aged grain from plants withhomozygous alleles of Line 9 or Line 22 had hexanal levels significantlyless than controls (P<0.05), whereas control lines all had increasedlevels of hexanal.

Example 8 Long Term Reduced Rancidity and Improved Shelf-Life of Lpx1Novel Alleles Up to the Equivalent of 24 Months at Room Temperature

In order to further test reduced rancidity and increased shelf-life ofwhole grain flour derived from grain of novel lipoxygenase mutantalleles, wheat Line 9 (Table 5) homozygous for the Lpx-D1(W494*) alleleand wheat Line 22 (Table 5) homozygous for both the Lpx-D1(W494*) andLpx-B1.2(W510*) alleles were milled as described in Example 5. Inaddition, sibling lines of either Lines 9 or 22 that were homozygouswild-type were grown at the same time and then milled and used ascontrols (wild-type siblings). The whole grain flours were subjected toaccelerated aging for 12 or 30 weeks at 37° C., equivalent to anestimated 10 or 24 months at room temperature, respectively. Hexanallevels were measured at Medallion Labs as described in Example 5.

As shown in FIG. 5, hexanal levels increased to 0.736 ppm in thewild-type sibling aged at 12 weeks. In contrast, hexanal values remainedunder the limit of detections of <0.3 ppm in wheat Lines 9 and 22 agedat 12 weeks. After 30 weeks of aging, hexanal values of Line 22wild-type sibling had much higher levels of 3.25 ppm. In even greatercontrast, hexanal values of wheat Line 9 aged at 30 weeks only increasedto 1.08 ppm and hexanal values of wheat Line 22 aged to 30 weeks onlyincreased to 0.691 ppm. This data demonstrates that novel alleles inLpx-D1 with reduced lipoxygenase activity singly and in combination withnovel alleles in Lpx-B1.2 significantly improve shelf-life of wholegrain flour.

Example 9 Reduced Production of Bitter Compound in Milled Whole GrainFlour and Dough Made from Grain of Lpx1 Novel Alleles

Oxidative degradation of free linoleic acid in whole wheat flour is themain contributor to the bitter taste in bread crumb (Bin & Peterson,Identification of bitter compounds in whole wheat bread crumb, FoodChemistry, 203:8-15 (2016)). The bitter compound9,12,13-trihydroxy-trans-10-octadecenoic (pinellic) acid is thought tobe a product of substrate epoxidation created during peroxidation bylipoxygenase enzymes (Gardner, Decomposition of linoleic acidhydroperoxides, JOAF 23:129-136 (1975)). To measure the improvement ofbitter taste in flour and dough made from grain of novel Lpx1 mutantalleles, whole grain samples were milled and stored for 6 months at 37°C. (equivalent to 19 months at room temperature). Fresh whole grainflour samples were milled immediately prior to analysis. Quantificationof 9,12,13-trihydroxy-trans-10-octadecenoic (pinellic) acid in flour anddough was performed using ultra-performance liquid chromatography tandemmass spectrometry (UPLC/MS/MS) at the Flavor Research and EducationCenter (University of Minnesota).

In a 15 mL reaction volume, 3 g of flour was extracted with anethanol-chloroform mix (75:25 v/v) spiked with a butyl 4-hydroxybenzoateat 100 mg/mL internal standard. Duplicate reactions were incubated on anorbital shaking table set to 120 rpm for 3 hours followed bycentrifugation at 8000 rpm for 15 minutes at 10° C. Aftercentrifugation, the supernatant was carefully separated from the organicphase, pooled in a 250 mL flat bottom flask, and concentrated via rotaryevaporation. The residue was re-solubilized in 4 mL of 10% methanol andthen subjected to solid phase extraction using a preconditioned 500 mgC18 cartridge (Supelco, Bellefonte, Pa., USA). Each sample was theneluted with 2 mL of methanol and filtered through 0.20 μm Nylon syringefilters (Millex, Billerica, Calif., USA) for further cleanup.

Dough was created by mixing 12 g of flour with 7.2 g of nanopure waterand then rested for 20 minutes to ensure adequate formation of pinellicacid. The dough was then divided into thirds by mass and each portionwas suspended in 20 mL of 75% (v/v) ethanol solution spiked with a butyl4-hydroxybenzoate (100 ng/mL) internal standard. The samples were thenincubated on an orbital shaking table set to 120 rpm for 3 hours,followed by centrifugation at 8000 rpm for 15 minutes at 10° C. 2 mL ofthe supernatant was subjected to solid phase extraction using apreconditioned 500 mg C18 cartridge (Supelco, Bellefonte, Pa., USA).Each sample was then eluted with 2 mL of methanol, and the pooledmixture was further diluted with 2 mL of nanopure water to preventsample breakthrough during mass spectral analysis.

Injections (2 μL) of flour or dough samples were separated on a 2.1mm×50 mm ACQUITY UPLC BEH C18 1.7 μm column (Waters, Milford, Mass.,USA) held at 25° C. MS conditions were as follows: ESI negative,desolvation temperature, 500° C.; source temperature, 120° C.; capillaryvoltage, 2.7 kV; desolvation gas, 700 L/hr; cone gas, 65 L/hr. UPLCmobile phase was maintained at a flow rate of 300 μL/min using a binarysolvent system of (A) 0.1% formic acid in ultrasonicated nanopure wateras the aqueous phase, and (B) 0.1% formic acid in acetonitrile for theorganic phase. The elution gradient started at 5% B (0-1 min), linearlyincreased to 50% B (1-6 min), then increased to 100% B (6-8 min) held at100% B (8-10 min), and re-equilibrated at 5% B (10-15 min). MS/MS iontransitions and collision energy were as follows:9,12,13trihydroxytrans-10-octadecenoicacid, ESI-329→211 (15 eV);butyl4hydroxybenzoate, ESI-193→136 (15 ev).

Whole grain flour milled from wheat Line 9 (Table 5) homozygous for theLpx-D1(W494*) allele and a sibling line that was wild-type for theLpx-D1 allele in Line 9 were aged for 6 months at 37° C. Freshly milledmaterial from these lines was also analyzed. In addition, freshly milledmaterial from wheat Line 22 (Table 5) homozygous for the Lpx-D1(W494*)and Lpx-B1.2(W510*) alleles and a sibling line wild-type for thesealleles was also analyzed.

As shown in FIG. 6, fresh milled whole grain flour made from wheat Line9 and Line 22 and their wild-type siblings had pinellic acidconcentrations of around 5 mg/kg flour. In whole grain flour aged for 6months, pinellic acid levels increased up to 20 mg/kg flour. However,Line 9 had a 15% decrease in pinellic acid formation compared to thewild-type sibling aged for the same length of time. This data shows thatnovel alleles in Lpx-D1 significantly reduces the production of bittercompounds in aged whole grain flour.

The effect of Lpx1 novel alleles on bitter compound formation in doughmade from whole grain is shown in FIG. 7. Dough made from freshly milledwhole grain flour from wheat Line 9 had a low pinellic acidconcentration of 60 mg/kg dough compared to the wild-type Line 9 siblingthat had over triple the amount of pinellic acid (200 mg/kg dough).Similarly, dough made from freshly milled whole grain flour of Line 22had a very low pinellic acid concentration of 32 mg/kg dough, while thewild-type Line 22 sibling had over ten times the amount of pinellic acid(340 mg/kg). This data demonstrates that dough made from whole grainflour from wheat with novel alleles in Lpx-D1 and in combination withnovel alleles in Lpx-B1.2 significantly reduces the concentration of themajor bitter compound found in whole grain products.

Also shown in FIG. 7, dough made from whole grain flour of wheat Line 9aged for 6 months had significantly reduced pinellic acid concentration(160 mg/kg of dough) compared to the wild-type sibling line hadconcentration at over six times this amount at 910 mg/kg doughdemonstrating improved shelf life of flour containing novel Lpx1alleles. This data shows that dough made from aged whole grain flourfrom wheat with novel alleles in Lpx-D1 significantly reduces theconcentration of the major bitter compound found in whole grainproducts.

The above examples are provided to illustrate the invention but notlimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims and all their equivalents. The examples above usedTILLING technology to create and identify mutations in one or more Lpxgenes of wheat but one of ordinary skill in the art would understandthat other methods such as targeted mutagenesis (also known assite-directed mutagenesis, site-specific mutagenesis,oligonucleotide-directed mutagenesis or genome editing) could be used tocreate the useful mutations disclosed herein in one or more Lpx1 loci ofwheat (see for example Zhang et al., PNAS 107(26):12028-12033, 2010;Saika et al., Plant Physiology 156:1269-1277, 2011). All publications,patents, and patent applications cited herein are hereby incorporated byreference.

Informational Sequence Listing SEQ ID NO Name Sequence Length 1Lpx-D1 Genomic Sequence 3863 bp 2 Lpx-D1 Coding Sequence 2586 bp 3Lpx-D1 Protein Sequence 862 amino acids 4 Lpx-B1.2 Genomic Sequence4263 bp 5 Lpx-B1.2 Coding Sequence 2586 bp 6 Lpx-B1.2 Protein Sequence862 amino acids SEQ ID NO: Lpx-D1 genomicATGATACTGGGCGGGCTCATCGACAGCCTGACCGGCGCG 1 nucleic acidAACAAGAACGCACGTCTCAAGGGCACGGCGGTGCTGATG sequenceAGGAAGAACGTGCTGGACCTCACCGACTTCGGCGCCACCATCATGGACGGCATCGGCGACTTCCTCGGCAAGGGCGTCACCTGCCAGCTTATCAGCTCCACCCTCATCGACCACGGTAAGCAGTGCACCCTTCTCCTCTTCCTCCTCCTCCTCTCCTCTCCTTACTAGATATGTCTTTTAATTTGTGTTGTCGGCCATGGATGCATGGATGTATCTCGATCGGCTAAAGATAGAGATAGCCTCGGTCGGTCGGTCGTCTTTAGCTGAGCATGGGCATGCCATGGAAAGAAGAGACAATAGTAGCATGGTGCGTGCACCAGAGCTTGCAGAGCATCGGATGCTCGAGACAAAGCAATAGAACAAGCAAGCACACGTCAAAAGTAACTATCACAACCTAAACTAAAGCTTTGAACTCGACTCCCAACAATCAATCAGGTTGACACGTACTAGTAAACTAAAGCACATGTGAGAACGAACGAACTGCGTGCGTGCGTGCAGACAACGGCGGGCGCGGGAAGGTGGGCGCGGAGGCGGAGCTGGAGCAGTGGGTGACGAGCCTGCCGTCGCTGACGACGGGGGAGTCCAAGTTCGGCCTCACCTTCGACTGGGAGGTGGAGAAGCTGGGGGTGCCCGGCGCCATCGTCGTCAACAACTACCACAGCTCCGAGTTCCTGCTCAAGACGGTCACCCTCCACGACGTCCCCGGCCGCGGCAACCTCTCCTTCGTCGCCAACTCCTGGATCTACCCCGCCGCCACCTACACCTACAGCCGCGTCTTCTTCGCCAACGATGTGAGTTGTGAGCCTCCCTTGTTTCCTCTCCTTTCCTTTCCATTTCACTGCCTTCGTCATTCATGGTCATTAAGTCTTCTTTGAGATAAGATAAGATTAGTAGGTGCAGAATTTATTCCGTGTTGGTAGAGAAAAAGGATATGGCTAGGTGCAGCAGAAGATTGAATGAAACCGGCACCGTGGCACCGTGGTAGGTGAAGAAAACTGTTGCCCTTGCCTGACCAAGTGTGCGACCTGCTGCTGCCGGGTTATTTCTTTGAGATAAGACACGTACGTGGGCTCACATGAACGCAAGCATGGCTCCACCACCATGGGACGACCTCGGTCGCTACATGGCCGCCTCAGAACTTTTAAAAGATGTTGCATGATACGGTAGTAGCACTCAATCCGGTTTACTTTGCCGAAACGGTGACATAAAACACATGAAAGAAAAAGCGATTATACTGCTCTAGTTGGCAAAGCAAAATCATCTAATTCACGTACTTCTTTTGTCATGAGCAAGCCATCGATCGGCTTCCGGCCTGCAGGTTCAGTGCTCGTCTAAAATGACAAATTTTCTTGCCATGTTACGCGCGTACAGACGTACCTGCCGAGCCAGATGCCGGCGGCGCTGAAGCCGTACCGCGACGACGAGCTCCGGAACCTGCGGGGCGACGATCGGCAGGGGCCCTACCAGGAGCACGACCGCGTCTACCGCTACGACGTCTACAACGACCTCGGCGAGGGCCGCCCGGTCCTCGGCGGCAGCGCCGAGCACCCTTACCCGCGGCGCGGCCGCACGGGCCGCAAGCCCAACGCCAGCGACCCGAGCCTGGAGAGCCGGCTGTCGCTGCTGGAGCAGATCTACGTGCCGCGGGACGAGAAGTTCGGCCACCTCAAGACGTCCGACTTCCTGGGCTACTCCATCAAGGCCATCACGCAGGGCATCCTGCCGGCGGTGCGCACCTACGTCGACACCACCCCCGGCGAGTTCGACTCCTTCCAGGACATCATGAACCTCTACGAGGGCGGCATCAAGCTGCCCATGGTGCCCGCCCTCGAGGAGCTGCGCAAGCAGTTCCCGCTCCAGCTCATCAAGGACCTGCTCCCCGTGGGCGGCGACTCGCTGCTGAAGCTCCCTGTGCCACATATCATCCAGGCGGACCAGCAGGCGTGGAGGACCGACGAGGAGTTCGCGCGCGAGGTGCTCGCCGGCGTCAACCCGGTCATGATCACGCGTCTCACGGTCAGTCAACGGTTACTATGTGTAGAATATGTATGTTGTCCATGGTGAGAGCACGCAAATCTTACTTGGTGTTGGGTCGGCATGCATGCAGGAGTTCCCGCCAAAAAGTAGTCTTGACCCTAGCAAGTTTGGTGACCACACCAGCACCATCACGGCGGCACACATCCAGAAGAACCTCGAGGGCCTCACCGTGCAGCAGGTAATAATATACACGATCGAGTTGGCCAACCCATCGCGATCAACTGTGATTTGGTGGGAGCAGGTCTAAGTAATTTTGGCTTGTTGCATGCAGGCGCTGGAAAGTAACAGGCTCTACATACTTGATCACCACGACCGGTTCATGCCGTTCCTGATCGAAGTCAACAACCTGCCCGGCAACTTCATCTACGCCACCAGGACCCTCTTCTTCCTGCGCGGCGACGGCAGGCTCACGCCGCTCGCCATCGAGCTGAGCGAGCCCGTCATCCTGGGCGGCCTCACCACCGCCAAGAGCAAGGTGTACACGCCGGTGCCGAGCGGCAGCGTCGAAGGCTGGGTGTGGGAGTTCGCCAAGGCCTACGTCGCCGTCAACGACTCCGGCTGGCACCAGCTCGTCAGCCACTGGTACGTGCACTACGGATTAACCAAACAATGGCGACACACCCCTCAAAAAAGAAAAGAAAAACAATGGCGACACTGACTCGGTGTGATTCAGTCAGTCGATGCACAACTGACCTATGATTGAAACGTGTAGGCTGAACACCCACGCGGTGATGGAGCCGTTTGTGATCTCGACGAACCGGCACCTCAGCGTGACGCACCCGGTGCACAAGCTGCTGAGCCCGCACTACCGCGACACCATGACCATCAACGCGCTGGCGCGGCAGACGCTCATCAACGCCGGCGGCATCTTCGAGATGACGGTGTTCCCGGGCAAGTTTGCGCTGGGGATGTCGTCGGTGGTGTACAAGGACTGGAAGTTCACCGAGCAGGGCCTGCCCGACGATCTCATCAAGAGGGGCATGGCGGTGGAGGACCTATCGAGCCCTTACAAGGTGCGGCTTCTGGTGTCGGACTACCCGTACGCGGCGGACGGGCTGGCGATCTGGCACGCCATCGAGCAGTACGTGGGCGAGTACCTGGCCATCTACTACCCGGACGACGGCGTGCTGCGGGGCGACACGGAGCTGCAGGCGTGGTGGAAGGAGGCGCGCGAGGTCGGGCACGGCGACCTCAAGGACGCGCCGTGGTGGCCGAGGATGCAGGGCGTGGGGGAGCTGGCCAAGGCGTGCACCACCATCATCTGGATCGGGTCGGCGCTGCACGCGGCGGTCAACTTCGGGCAGTACCCGTACGCGGGGTTCCTCCCGAACCGGCCGACGGTGAGCCGGCGCCGCATGCCGGAGCCCGGGACGGACGCGTACGCGGAGCTGGAGCGCGACCCGGAGCGGGCCTTCATCCACACCATCACCAGCCAGATCCAGACCATCATCGGCATCTCGCTGTTGGAGGTGCTGTCCAAACACTCCTCTGACGAGCTCTACCTGGGGCAGCGCGACACGCCGGAGTGGACCTCGGACCCCAAGGCCCTGGAGGTGTTCAAGCGGTTCAGCGAGCGGCTGGTGGAGATCGAGAGCAAGGTGGTGGGCATGAACCACGACCCGCAGCTGTTGAACCGCAACGGCCCGGCCAAGCTCCCCTACATGCTGCTCTACCCCAACACCTCCGACCACAAGGGCGCCGCCGCCGGCCTCACCGCCAAGGGCATCCCCAACAGCATCTCCATC TGA SEQ ID NO: Lpx-D1 CodingATGATACTGGGCGGGCTCATCGACAGCCTGACCGGCGCG 2 sequenceAACAAGAACGCACGTCTCAAGGGCACGGCGGTGCTGATGAGGAAGAACGTGCTGGACCTCACCGACTTCGGCGCCACCATCATGGACGGCATCGGCGACTTCCTCGGCAAGGGCGTCACCTGCCAGCTTATCAGCTCCACCCTCATCGACCACGACAACGGCGGGCGCGGGAAGGTGGGCGCGGAGGCGGAGCTGGAGCAGTGGGTGACGAGCCTGCCGTCGCTGACGACGGGGGAGTCCAAGTTCGGCCTCACCTTCGACTGGGAGGTGGAGAAGCTGGGGGTGCCCGGCGCCATCGTCGTCAACAACTACCACAGCTCCGAGTTCCTGCTCAAGACGGTCACCCTCCACGACGTCCCCGGCCGCGGCAACCTCTCCTTCGTCGCCAACTCCTGGATCTACCCCGCCGCCACCTACACCTACAGCCGCGTCTTCTTCGCCAACGATACGTACCTGCCGAGCCAGATGCCGGCGGCGCTGAAGCCGTACCGCGACGACGAGCTCCGGAACCTGCGGGGCGACGATCGGCAGGGGCCCTACCAGGAGCACGACCGCGTCTACCGCTACGACGTCTACAACGACCTCGGCGAGGGCCGCCCGGTCCTCGGCGGCAGCGCCGAGCACCCTTACCCGCGGCGCGGCCGCACGGGCCGCAAGCCCAACGCCAGCGACCCGAGCCTGGAGAGCCGGCTGTCGCTGCTGGAGCAGATCTACGTGCCGCGGGACGAGAAGTTCGGCCACCTCAAGACGTCCGACTTCCTGGGCTACTCCATCAAGGCCATCACGCAGGGCATCCTGCCGGCGGTGCGCACCTACGTCGACACCACCCCCGGCGAGTTCGACTCCTTCCAGGACATCATGAACCTCTACGAGGGCGGCATCAAGCTGCCCATGGTGCCCGCCCTCGAGGAGCTGCGCAAGCAGTTCCCGCTCCAGCTCATCAAGGACCTGCTCCCCGTGGGCGGCGACTCGCTGCTGAAGCTCCCTGTGCCACATATCATCCAGGCGGACCAGCAGGCGTGGAGGACCGACGAGGAGTTCGCGCGCGAGGTGCTCGCCGGCGTCAACCCGGTCATGATCACGCGTCTCACGGAGTTCCCGCCAAAAAGTAGTCTTGACCCTAGCAAGTTTGGTGACCACACCAGCACCATCACGGCGGCACACATCCAGAAGAACCTCGAGGGCCTCACCGTGCAGCAGGCGCTGGAAAGTAACAGGCTCTACATACTTGATCACCACGACCGGTTCATGCCGTTCCTGATCGAAGTCAACAACCTGCCCGGCAACTTCATCTACGCCACCAGGACCCTCTTCTTCCTGCGCGGCGACGGCAGGCTCACGCCGCTCGCCATCGAGCTGAGCGAGCCCGTCATCCTGGGCGGCCTCACCACCGCCAAGAGCAAGGTGTACACGCCGGTGCCGAGCGGCAGCGTCGAAGGCTGGGTGTGGGAGTTCGCCAAGGCCTACGTCGCCGTCAACGACTCCGGCTGGCACCAGCTCGTCAGCCACTGGCTGAACACCCACGCGGTGATGGAGCCGTTTGTGATCTCGACGAACCGGCACCTCAGCGTGACGCACCCGGTGCACAAGCTGCTGAGCCCGCACTACCGCGACACCATGACCATCAACGCGCTGGCGCGGCAGACGCTCATCAACGCCGGCGGCATCTTCGAGATGACGGTGTTCCCGGGCAAGTTTGCGCTGGGGATGTCGTCGGTGGTGTACAAGGACTGGAAGTTCACCGAGCAGGGCCTGCCCGACGATCTCATCAAGAGGGGCATGGCGGTGGAGGACCTATCGAGCCCTTACAAGGTGCGGCTTCTGGTGTCGGACTACCCGTACGCGGCGGACGGGCTGGCGATCTGGCACGCCATCGAGCAGTACGTGGGCGAGTACCTGGCCATCTACTACCCGGACGACGGCGTGCTGCGGGGCGACACGGAGCTGCAGGCGTGGTGGAAGGAGGCGCGCGAGGTCGGGCACGGCGACCTCAAGGACGCGCCGTGGTGGCCGAGGATGCAGGGCGTGGGGGAGCTGGCCAAGGCGTGCACCACCATCATCTGGATCGGGTCGGCGCTGCACGCGGCGGTCAACTTCGGGCAGTACCCGTACGCGGGGTTCCTCCCGAACCGGCCGACGGTGAGCCGGCGCCGCATGCCGGAGCCCGGGACGGACGCGTACGCGGAGCTGGAGCGCGACCCGGAGCGGGCCTTCATCCACACCATCACCAGCCAGATCCAGACCATCATCGGCATCTCGCTGTTGGAGGTGCTGTCCAAACACTCCTCTGACGAGCTCTACCTGGGGCAGCGCGACACGCCGGAGTGGACCTCGGACCCCAAGGCCCTGGAGGTGTTCAAGCGGTTCAGCGAGCGGCTGGTGGAGATCGAGAGCAAGGTGGTGGGCATGAACCACGACCCGCAGCTGTTGAACCGCAACGGCCCGGCCAAGCTCCCCTACATGCTGCTCTACCCCAACACCTCCGACCACAAGGGCGCCGCCGCCGGCCTCACCGCCAAGGGCATCCCCAACAGCATCTCCATCTGA SEQ ID Lpx-D1 AminoMILGGLIDSLTGANKNARLKGTAVLMRKNVLDLTDFGATIM NO:3 acid sequenceDGIGDFLGKGVTCQLISSTLIDHDNGGRGKVGAEAELEQWVTSLPSLTTGESKFGLTFDWEVEKLGVPGAIVVNNYHSSEFLLKTVTLHDVPGRGNLSFVANSWIYPAATYTYSRVFFANDTYLPSQMPAALKPYRDDELRNLRGDDRQGPYQEHDRVYRYDVYNDLGEGRPVLGGSAEHPYPRRGRTGRKPNASDPSLESRLSLLEQIYVPRDEKFGHLKTSDFLGYSIKAITQGILPAVRTYVDTTPGEFDSFQDIMNLYEGGEKLPMVPALEELRKQFPLQLIKDLLPVGGDSLLKLPVPHIIQADQQAWRTDEEFAREVLAGVNPVMITRLTEFPPKSSLDPSKFGDHTSTITAAHIQKNLEGLTVQQALESNRLYILDHHDRFMPFLIEVNNLPGNFIYATRTLFFLRGDGRLTPLAIELSEPVILGGLTTAKSKVYTPVPSGSVEGWVWEFAKAYVAVNDSGWHQLVSHWLNTHAVMEPFVISTNRHLSVTHPVHKLLSPHYRDTMTINALARQTLINAGGEFEMTVFPGKFALGMSSVVYKDWKFTEQGLPDDLIKRGMAVEDLSSPYKVRLLVSDYPYAADGLAIWHAIEQYVGEYLAIYYPDDGVLRGDTELQAWWKEAREVGHGDLKDAPWWPRMQGVGELAKACTTIIWIGSALHAAVNFGQYPYAGFLPNRPTVSRRRMPEPGTDAYAELERDPERAFIHTITSQIQTIIGISLLEVLSKHSSDELYLGQRDTPEWTSDPKALEVFKRFSERLVEIESKVVGMNHDPQLLNRNGPAKLPYMLLYPNTSDHKGAAAGLTAKGIPNSISI SEQ ID NO: Lpx-B1.2 genomicATGATACTGGGCGGGCTCGTCGACAGCCTGACCGGCGCG 4 nucleic acidAACAAGAGCGCACGGCTCCAGGGCACGGTGGTGCTCATG sequenceAGGAAGAACGTGCTGGACCTCAACGACTTCGGCGCCACCATCATGGACGGCATCGGCGAGTTCATCGGCAAGGGCGTCACCTGCCAGCTTATCAGCTCCACCCTCGTCGACCACGGTAAGCAGTGCACCATTCTCCTCTTCCTCCTCCTCCTCTCCTTGGTAGATATTTCTTTTGTGTTGTCGGCCATGGATGCATGGATGTATCTCGATCGGCTAAAGAATGATAGATAGATAGCCATGGTCGGTCGTCTTTAGCTGAGCATGGGCATGGAAAGAAGAGACGAGAGCATGGTGCGTGCACCAGAGCTTACAGAGCACCAGATGCTCCAGACAAAGCAATAGAACAAGCAAGGACACGTCGCCAAAAGCAACAAACACAACCTAAACTAAAGCACAAAGACGTAGCGATGAAAAAAGCATCGTGGGCAGATGCTCTAACCATGCGAGACCGTGCTCCGTGCAGACAACGGCGGGCGTGGGAAGGTGGGCGCGGAGGCGGAGCTGGAGCAGTGGGTGACGAGCCTGCCGTCGCTGACGACGGGGGAGTCCAAGTTCGGCCTCACCTTCGACTGGGAGGTGGAGAAGCTGGGCGTGCCGGGCGCCATCATCGTCAACAACCACCACAGCTCCGAGTTCCTGCTCAAGACCGTCACCCTCCACGACGTCCCCGGCCGCGGCAACCTCTCCTTCGTCGCCAACTCCTGGATCTACCCCGTTGGCAGCTACACCTACAGCCGCGTCTTCTTCGCCAACGATGTGAGTTGTGACTTGTGAGCCTTGCCTTTCCTCTCCTTTCCTTTTCACTGGCTTCCTCATTCATGGTCATTTAAGTCTTCTCTGAGATAAGATAAGATTAGTAGGTGCAGAATTTATTCCATGTTGGTAGAAAAAAGATATGGCTAGGTGCAGCAGAAGATTGAATGAATGTGGCACCGTGGTTGGTGAAGACAACTGCTGCCCTTGACTGACCTGCTGCTGGGTTCTTCCTTTGGGATAAGAACACCGAGCGAGACACGTACGTACGTGAGCTCAAACGAACCCATGGCTCCACCTCCATGACCTGATCCTTCCCTTGAAACGACCTAAGATAGTTACATGGCCGAGCCCAGAACAAACTTTTAAAAAGAGATGCTGCATAGTCATGATACAGTGACATAATAAAACACATGAAAGAAGAGGCGATTATTGCTCTAGTTGGCAAAGCAAAATAATCTACTAACTCTTGTGTAGTACTACTAGCTAGCAACATACGTACGGGAGTTCTTTTGTCATAAACAAGCGATCGATCGGCTTCCTGCAGGTTCAGTGCTCATCTAAAATGACAAATTTTTTGGTATGTGTACCTACGCGCAGACGTACCTGCCGAGCCAGATGCCAGCGGCGCTGAAGCCGTACCGCGACGACGAGCTCCGGAACCTGCGGGGCGACGACCGGCAGGGCCCCTACCAGGAGCACGACCGCGTCTACCGCTACGACGTCTACAACGACCTCGGCGAGGGCCGCCCCGTCCTCGGCGGCAGCGCCGAGCACCCCTATCCGCGCCGCGGCCGCACCGGGCGCAAGCCCAACGCCAACGACCCGAGCTTGGAGAGCCGGCTGTCGCTGCTGGAGCAGATCTACGTGCCGCGGGACGAGAAGTTCGGCCACCTCAAGACGTCCGACTTCCTGGGCTACTCCATCAAGGCCATCACGCAGGGCATCCTGCCGGCGGTGCGCACCTACGTCGACACCACCCCCGGCGAGTTCGACTCCTTCCAGGACATCATCAACCTCTACGAGGGCGGCATCAAGCTGCCCAACGTCCCCGCCCTCGAGGAGCTGCGCAAGCAGTTCCCGCTCCAGCTCATCAAGGACCTCCTCCCCGTGGGTGGCGACTCGCTGCTCAAGCTCCCCGTCCCCCACATCATCCAGGCGGACCAGCAGGCGTGGCGGACCGACGAGGAGTTCTCCCGGGAGGTCCTTGCCGGCGTCAACCCGGTCATGATCACGCGTCTCACGGTGAGTCAACAATAATTGAACAGTCTTACTAAAGGCCCGTTCGGAGGCTCTCCACTCCTCAACTCTCTCCCGGAGCGGCCGGAGCTTCAGTTTAAAATTATGGAGTGGCCGAAGAGGTACTCCGCAGATCCTTGTATTCTGCGGAGCTGGGCCAGTGCCGAACAGGGCCTAAGTCTCAGTCGATCTATATCCGACAGATCTTACATTAAGATTCTTTTCAGTTTTTCTTTTTCTTTTTTTGCATGTTATATCAAATTTGACTAAGACTTCATTAAATCTCGGTCGACAGAAACTTAGCCACACACCATAATTGAACGATGAATGAGTATGCTATCCATGGATCGAGAACCGAGAGGTGAGAGCGTGCCTGATCTTAATTTGTGTTGGGTGGCATGCATACAGGAGTTCCCGCCAAAAAGTAGTCTGGACCCTAGCAAGTTTGGTGACCACACCAGCACCGTCACGGCGGCGCACATCGAGAAAAACCTCGAAGGCCTCACCGTGCAGCAGGTAATAATACTACAATACACGAGTCGGCCAACCCATCGCGATCAACTGTGATTTGATGGAAGCAGGTGTAACTAATTTTGGCATGTTGCAACTTGTTGCATGCAGGCCCTTGAAAGCAACCGGTTGTACATCCTTGATCACCACGACCGGTTCATGCCGTTCCTCATCGACGTCAACAACCTGCCCGGCAACTTCATCTACGCCACGAGGACCCTCTTCTTCCTGCGCGGCGACGGCAGGCTCACGCCGCTCGCCATCGAGCTCAGCGAGCCTGTCATACAGGGCGGCCTCACCACCGCCAAGAGCAAGGTGTACACGCCGGTGCCGAGCGGCAGCGTCGAAGGATGGGTGTGGGAGTTCGCCAAGGCCTACGTCGCCGTCAACGACTCTGGGTGGCACCAGCTCGTCAGCCACTGGTACGTGCACTACGGACTAACCAAACAACTGAGAACAGTCTTACTAAGTCTCAGTCGATCTATATCCGACACTGACTCGGTGTGATTCAGTCAGTCGATGCACAACTGACCTATGATTGAAACGTGCAGGCTGAACACTCATGCGGTGATGGAGCCGTTTGTGATCTCGACGAACCGGCAGCTCAGCGTGACGCACCCGGTGCACAAGCTGCTGAGCCCGCACTACCGCGACACGATGACCATCAACGCGCTAGCGCGGCAGACGCTCATCAACGCCGGCGGCATCTTCGAGATGACGGTGTTCCCGGGCAAGTTCGCGTTGGGGATGTCGTCAGTGGTGTACAAGGACTGGAAGTTCACGGAGCAGGGCCTGCCCGACGATCTCATCAAGAGGTACGTACCAAGTATAATGTACTAATGAAACTGTGTTACAAATCATGCTTTTAGATGACTGACGACACATACGTGGTGCATAACAAAAAAATGCAGGGGCATGGCGGTGGAGGACCCGTCGAGCCCGTACAAGGTGAGGCTGCTGGTGTCTGACTACCCGTACGCGGCGGACGGGCTGGCGATCTGGCACGCCATCGAGCAGTACGTGAGCGAGTACCTGGCCATTTACTACCCGAACGATGGCGTGGTGCAGGGCGACGTGGAGCTGCAGGCGTGGTGGAAGGAGGTGCGCGAGGTGGGGCACGGCGACCTCAAGGTCGCGCCATGGTGGCCGAGGATGCAAGCCGTGGGCGAGCTGGCCAAGGCGTGCACCACCATCATCTGGATCGGGTCGGCGCTGCATGCGGCGGTCAACTTCGGGCAGTACCCATACGCGGGGTTCCTCCCGAACCGGCCGACGGTGAGCCGGCGCCGCATGCCGGAGCCGGGGACCGAGCAGTACGCGGAGCTGGAGCGCGACCCGGAGCGGGCCTTCATCCACACCATCACTAGCCAGATCCAGACCATCATCGGCATCTCGCTGCTGGAGGTGCTGTCGAAGCACTCCTCCGACGAGCTCTACCTCGGGCAGCGTGACACGCCGGAGTGGACCTCGGACCCCAAGGCCCTGGAGGTGTTCAAGCGGTTCAGCGAGCGGCTAGCGGAGATCGAGAGCAAGGTGGTGGGCATGAACCACGACCCGCAGCTGTTGAACCGCAACGGTCCGGCCAAGTTCCCCTACATGTTGCTCTACCCCAACACCTCCGATCACAAGGGCGCCGCCGCCGGGCTCACCGCTAAGGGCATCCCCAACAGCATCTCCATC TGA SEQ ID NO: Lpx-B1.2 CodingATGATACTGGGCGGGCTCGTCGACAGCCTGACCGGCGCG 5 sequenceAACAAGAGCGCACGGCTCCAGGGCACGGTGGTGCTCATGAGGAAGAACGTGCTGGACCTCAACGACTTCGGCGCCACCATCATGGACGGCATCGGCGAGTTCATCGGCAAGGGCGTCACCTGCCAGCTTATCAGCTCCACCCTCGTCGACCACGACAACGGCGGGCGTGGGAAGGTGGGCGCGGAGGCGGAGCTGGAGCAGTGGGTGACGAGCCTGCCGTCGCTGACGACGGGGGAGTCCAAGTTCGGCCTCACCTTCGACTGGGAGGTGGAGAAGCTGGGCGTGCCGGGCGCCATCATCGTCAACAACCACCACAGCTCCGAGTTCCTGCTCAAGACCGTCACCCTCCACGACGTCCCCGGCCGCGGCAACCTCTCCTTCGTCGCCAACTCCTGGATCTACCCCGTTGGCAGCTACACCTACAGCCGCGTCTTCTTCGCCAACGATACGTACCTGCCGAGCCAGATGCCAGCGGCGCTGAAGCCGTACCGCGACGACGAGCTCCGGAACCTGCGGGGCGACGACCGGCAGGGCCCCTACCAGGAGCACGACCGCGTCTACCGCTACGACGTCTACAACGACCTCGGCGAGGGCCGCCCCGTCCTCGGCGGCAGCGCCGAGCACCCCTATCCGCGCCGCGGCCGCACCGGGCGCAAGCCCAACGCCAACGACCCGAGCTTGGAGAGCCGGCTGTCGCTGCTGGAGCAGATCTACGTGCCGCGGGACGAGAAGTTCGGCCACCTCAAGACGTCCGACTTCCTGGGCTACTCCATCAAGGCCATCACGCAGGGCATCCTGCCGGCGGTGCGCACCTACGTCGACACCACCCCCGGCGAGTTCGACTCCTTCCAGGACATCATCAACCTCTACGAGGGCGGCATCAAGCTGCCCAACGTCCCCGCCCTCGAGGAGCTGCGCAAGCAGTTCCCGCTCCAGCTCATCAAGGACCTCCTCCCCGTGGGTGGCGACTCGCTGCTCAAGCTCCCCGTCCCCCACATCATCCAGGCGGACCAGCAGGCGTGGCGGACCGACGAGGAGTTCTCCCGGGAGGTCCTTGCCGGCGTCAACCCGGTCATGATCACGCGTCTCACGGAGTTCCCGCCAAAAAGTAGTCTGGACCCTAGCAAGTTTGGTGACCACACCAGCACCGTCACGGCGGCGCACATCGAGAAAAACCTCGAAGGCCTCACCGTGCAGCAGGCCCTTGAAAGCAACCGGTTGTACATCCTTGATCACCACGACCGGTTCATGCCGTTCCTCATCGACGTCAACAACCTGCCCGGCAACTTCATCTACGCCACGAGGACCCTCTTCTTCCTGCGCGGCGACGGCAGGCTCACGCCGCTCGCCATCGAGCTCAGCGAGCCTGTCATACAGGGCGGCCTCACCACCGCCAAGAGCAAGGTGTACACGCCGGTGCCGAGCGGCAGCGTCGAAGGATGGGTGTGGGAGTTCGCCAAGGCCTACGTCGCCGTCAACGACTCTGGGTGGCACCAGCTCGTCAGCCACTGGCTGAACACTCATGCGGTGATGGAGCCGTTTGTGATCTCGACGAACCGGCAGCTCAGCGTGACGCACCCGGTGCACAAGCTGCTGAGCCCGCACTACCGCGACACGATGACCATCAACGCGCTAGCGCGGCAGACGCTCATCAACGCCGGCGGCATCTTCGAGATGACGGTGTTCCCGGGCAAGTTCGCGTTGGGGATGTCGTCAGTGGTGTACAAGGACTGGAAGTTCACGGAGCAGGGCCTGCCCGACGATCTCATCAAGAGGGGCATGGCGGTGGAGGACCCGTCGAGCCCGTACAAGGTGAGGCTGCTGGTGTCTGACTACCCGTACGCGGCGGACGGGCTGGCGATCTGGCACGCCATCGAGCAGTACGTGAGCGAGTACCTGGCCATTTACTACCCGAACGATGGCGTGGTGCAGGGCGACGTGGAGCTGCAGGCGTGGTGGAAGGAGGTGCGCGAGGTGGGGCACGGCGACCTCAAGGTCGCGCCATGGTGGCCGAGGATGCAAGCCGTGGGCGAGCTGGCCAAGGCGTGCACCACCATCATCTGGATCGGGTCGGCGCTGCATGCGGCGGTCAACTTCGGGCAGTACCCATACGCGGGGTTCCTCCCGAACCGGCCGACGGTGAGCCGGCGCCGCATGCCGGAGCCGGGGACCGAGCAGTACGCGGAGCTGGAGCGCGACCCGGAGCGGGCCTTCATCCACACCATCACTAGCCAGATCCAGACCATCATCGGCATCTCGCTGCTGGAGGTGCTGTCGAAGCACTCCTCCGACGAGCTCTACCTCGGGCAGCGTGACACGCCGGAGTGGACCTCGGACCCCAAGGCCCTGGAGGTGTTCAAGCGGTTCAGCGAGCGGCTAGCGGAGATCGAGAGCAAGGTGGTGGGCATGAACCACGACCCGCAGCTGTTGAACCGCAACGGTCCGGCCAAGTTCCCCTACATGTTGCTCTACCCCAACACCTCCGATCACAAGGGCGCCGCCGCCGGGCTCACCGCTAAGGGCATCCCCAACAGCATCTCCATCTGA SEQ ID Lpx-B1.2 AminoMILGGLVDSLTGANKSARLQGTVVLMRKNVLDLNDFGATI NO:6 acidsMDGIGEFIGKGVTCQLISSTLVDHDNGGRGKVGAEAELEQW sequenceVTSLPSLTTGESKFGLTFDWEVEKLGVPGAIIVNNHHSSEFLLKTVTLHDVPGRGNLSFVANSWIYPVGSYTYSRVFFANDTYLPSQMPAALKPYRDDELRNLRGDDRQGPYQEHDRVYRYDVYNDLGEGRPVLGGSAEHPYPRRGRTGRKPNANDPSLESRLSLLEQIYVPRDEKFGHLKTSDFLGYSIKAITQGILPAVRTYVDTTPGEFDSFQDIINLYEGGIKLPNVPALEELRKQFPLQUKDLLPVGGDSLLKLPVPHIIQADQQAWRTDEEFSREVLAGVNPVMITRLTEFPPKSSLDPSKFGDHTSTVTAAHIEKNLEGLTVQQALESNRLYILDHHDRFMPFLIDVNNLPGNFIYATRTLFFLRGDGRLTPLAIELSEPVIQGGLTTAKSKVYTPVPSGSVEGWVWEFAKAYVAVNDSGWHQLVSHWLNTHAVMEPFVISTNRQLSVTHPVHKLLSPHYRDTMTINALARQTLINAGGEFEMTVFPGKFALGMSSVVYKDWKFTEQGLPDDLIKRGMAVEDPSSPYKVRLLVSDYPYAADGLAIWHAIEQYVSEYLAIYYPNDGVVQGDVELQAWWKEVREVGHGDLKVAPWWPRMQAVGELAKACTTIIWIGSALHAAVNFGQYPYAGFLPNRPTVSRRRMPEPGTEQYAELERDPERAFIHTITSQIQTIIGISLLEVLSKHSSDELYLGQRDTPEWTSDPKALEVFKRFSERLAEIESKVVGMNHDPQLLNRNGPAKFPYMLLYPNTSDHKGAAAGLTAKGEPNSISI SEQ ID NO: TaLpx1D_4_L-1TGGTGAGAGCACGCAAATCTTACTTGG 7 SEQ ID NO: TaLpxB1.2-D1-5R1CGTTTCAATCATAGGTCAGTTGTGCATCGA 8 SEQ ID NO: TaLpx1D_67 R 3CGCGTACGGGTAGTCCGACACCAGAAG 9 SEQ ID NO: TaLpx1D_In1_L4GCATGCCATGGAAAGAAGAGACAATAGTAGC 10 SEQ ID NO: TaLpx1D_3_R-1TGCGTGCTCTCACCATGGACAACATACATA 11 SEQ ID NO: TaLpx1D_Ex5_L6GCAGGCGCTGGAAAGTAACAGGCTCT 12 SEQ ID NO: TaLpx1D_Ex7_R3TGGACGAGACGAAGCTCCGATGTACCA 13 SEQ ID NO: TaLpx1.2B_4_L-1GAGGTGAGAGCGTGCCTGATCTTAATTTG 14 SEQ ID NO: LpxD1proLTCATGCCGCTGATCGTCGC 16 SEQ ID NO: LpxD1proR CTTGCTGCTATTTCAGTACCG 17SEQ ID Lpx-D1 Promoter CACCGCTGTAACAGCCCCATTGTCTAGCTCAAAGAACTCT NO: 15and Exon1 CTCATGGCCACAATTACATCAGGAGGAAGCTGCTCTTTGAACTTTACAGCGAAGGCATCCAGGGTGGCAGTGGTGACGTCCTGCCCGTTCTTGATGATGCCCAGGCTGCGGCCAATAAACTGCTCAGCCGCCTGTGCCGCCGGCGTCGTTGCTGCGAGGACAGCAGGGCGCCTGATCCTCTGCAGGGAGAGGCCACCCACCTTGGAGATCTTCACGCCAGCGAGAGTTTTCCGACGGGCTGCCGGCTTCCTGGGTGGTGTTGAAGCAGGGGAAGGAGGGAGATCCGGGGCAGACATCAAGAGCGCAGGCTGGCGAGCACAGAACAGTGGTTCCGGGAGAGCCACCCCCAGGCTTGGCACAAGCAGCTCAGCAAGCCCCGCCGGCAACACGTCATCATCTGCACGTTTACGAGCCCGTGGAGATGTCGGGGTTCGCTGCAGGGGGCCACTGCTGAGGACGGGTGGTGTAGGAGATGCCTCAAAGCCGGGAGGGCGTACTGGCGAGACGTGGAGGAGGGGGCTCAACATGGAGACCGGCGAGCAAAGCTCAATGAGTGCTGGACTCCTCTCTTGAATGGCCTCTGTAGCAACCCTAGCCTCATCACGCTTGGAGGGGGGTGGTGAGCGGAGCACGGACTTGGGGGAGAGGGGCGGCGAAGCCGGAGGAGTGCGGGCGTCCCTTGAGTCCCGCCTGGCACTGCCTGGGCGCGCGTGCCTATACGGGCTGCGACCACGAGCCTCCACCGGAGCAGCCAGAGCGGTGGCCGGAGGGGCTCGACCCATCTCCAGGGCGCAAGCCTCGGGGTCGCAGACTGCGCCGGAGCCGAAGAGGAGAGGGGCGGCGCCGGAGCCCAAGAGGACAGGGGCGGCGGCCGGCGGGGGCGGCGGCGATGCAGCGCGCCTGCGGCCGTCGCGGTTGCCGCGATGGTCATCCCTGCTTCCCCTGTCTTGGTCATGCCGCTGATCGTCGCGTGGCGCACGGGAAAGGCTCCTGTGGACACGTGCACCGCTGTCTTGACCTCCCCTGGACGCGTCGCGCCCGTCGCGGCCACGACCGCGGTCACCATCTTCATCTCGATCGCGCCGATCCTGACGCACGGGACAAGAGACGCGCTCACGCCTGTCGCGAGCTTTGGCCTCGCCATCCACGATGTTGTAGCGCCACGTCTCGGTCGGCGGACTATTCACACAACCGATGCGTCGCCGCTCTCGGCCCGCGTGCATGTATGGCGCGCCCCGCCGCTCGCGGACGCGTACGTTTGGCCGCCAAATCCACCGGACGCAAAGATATATCCTCGTCGTAGCGACGGAAGGACGCTTGCATGTATGGATGGAGAAGCCGGTGAAACAAAAGGGAAAACAAGATGGACGCCGCCGTCTCGCCAATCTCGCCCACCCACACTAGATCCCGGCGCTCCATGGAGACTTGGGGAAGACACGTCCCCCAACTCTATAATGCCGCCTCCAGCGCCGCCGTGGAGGAACCGGAGTTGCGGAAGATGTATTGGACACTAGTGCGCGGCCACCACCCGGACGAAGCCAAACGAAAACCTAACCCTACAGCTATCTACAAACGGAATGCATGCACCACTCGGCCAACTATTCACACGACCAACGCGTCGCCGCTCTCGGTCCGCATGTATGTATGGCGCGCCCCGCCGGTCGCCGACGCGTACGTTTGACCGCCAAATGCACCGGACGCAAAAATATGTCCTCGTCGCAGCGACGTGCGCGGATGCCCCATCCCTGGAGCTGGACCGCGCCATGCGAAAGACGAGCCGGGCGCGTCGCCGTCCGGCGCTGCGGGTAGTCCGATCCGATCGAGCCAGCCGCTACGCGCCGGCGCCGCTTGCAGCAGAAAAGGACGGGGCGATGGATCCATCGCAACAAGCGCGGGCAGGCGCCACGCCATCCACGTAACAGCCAGGCCAAGAAAACTCGTGTACGAAGCTCCGTGCTCAGCGCTGGGCACGCGCGCGCTCTCGCCGCACCCCCACCCCCTATAAATTGGCCGGCCCGCGCTGCGACCTCCTCACACGCTTTCCCTCACACAACACACACCCATCTCCTTCCGCACAGCTCTCCACCGAAAGGCACTGGTAGTGCAGTTGAAGTAGCGACGGTACTGAAATAGCAGCAAGATGATACTGGGCGGGCTCATCGACAGCCTGACCGGCGCGAACAAGAACGCACGTCTCAAGGGCACGGCGGTGCTGATGAGGAAGAACGTGCTGGACCTCACCGACTTCGGCGCCACCATCATGGACGGCATCGGCGACTTCCTCGGCAAGGGCGTCACCTGCCAGCTTATCAG CTCCACCCTCATCGACCACG

What is claimed is:
 1. A wheat plant comprising at least one mutation inan Lpx1 gene in at least one of a B or D genome, wherein the Lpx1 geneof the B genome is Lpx-B1.2, and the at least one mutation in theLpx-B1.2 gene comprises a tryptophan substitution to a stop codon atamino acid position 510 (W510*) of SEQ ID No. 6, or a tryptophansubstation to a stop codon at amino acid position 494 (W494*) of SEQ IDNo. 6, or a guanine substitution to an adenine at nucleotide position2691 of SEQ ID No. 4, and further wherein the Lpx1 gene of the D genomeis Lpx-D1, and at least one mutation in the Lpx-D1 gene comprises atryptophan substitution to a stop codon at amino acid position 494(W494*) of SEQ ID No. 3, or a tryptophan substitution to a stop codon atamino acid position 81 (W81*) of SEQ ID No. 3, or a tryptophansubstitution to a stop codon at amino acid position 101 (W101*) of SEQID No. 3, or a tryptophan substitution to a stop codon at amino acidposition 517 (W517*) of SEQ ID No. 3, or a guanine substitution to anadenine at nucleotide position 1538 of SEQ ID No.
 15. 2. The wheat plantof claim 1 comprising at least one mutation in the B genome of theLpx-B1.2 gene, wherein the at least one mutation in the Lpx-B1.2 genecomprises a tryptophan substitution to a stop codon at amino acidposition 510 (W510*) of SEQ ID No.
 6. 3. Wheat grain from the wheatplant of claim
 2. 4. Flour comprising wheat grain of claim
 3. 5. Thewheat plant of claim 1 comprising at least one mutation in the D genomeof the Lpx-D1 gene, wherein the at least one mutation in the Lpx-D1 genecomprises a tryptophan to a stop codon at amino acid position 494(W494*) of SEQ ID No.
 3. 6. Wheat grain from the wheat plant of claim 5.7. Flour comprising wheat grain of claim
 6. 8. Wheat grain from thewheat plant of claim
 1. 9. Flour comprising wheat grain of claim
 8. 10.A wheat seed, plant part or progeny thereof from a wheat plant of claim1, wherein said progeny comprises the Lpx1 mutation or mutations. 11.The wheat plant of claim 1, wherein milled grain from said wheat planthas a property selected from the group consisting of: (a) increasedshelf-life; (b) increased oxidative stability; (c) decreased productionof Lpx1 protein; (d) decreased activity of the Lpx1 protein; (e)decreased hexanal production; (f) decreased pinellic acid production;(g) decreased decomposition products from fatty acids; or (h) improvedsensory characteristics as compared to milled grain from a wild typewheat plant.
 12. A wheat plant comprising at least one mutation in anLpx1 gene in each of a B and a D genome, wherein the Lpx1 gene of the Bgenome is Lpx-B1.2, and the at least one mutation in the Lpx-B1.2 genecomprises a tryptophan substitution to a stop codon at amino acidposition 510 (W510*) of SEQ ID No. 6, or a tryptophan substitution to astop codon at amino acid position 494 (W494*) of SEQ ID No. 6, or aguanine substitution to an adenine at nucleotide position 2691 of SEQ IDNo. 4, and further wherein the Lpx1 gene of the D genome is Lpx-D1, andthe at least one mutation in the Lpx-D1 gene comprises a tryptophansubstitution to a stop codon at amino acid position 494 (W494*) of SEQID No. 3, or a tryptophan substitution to a stop codon at amino acidposition 81 (W81*) of SEQ ID No. 3, or a tryptophan substitution to astop codon at amino acid position 101 (W101*) of SEQ ID No. 3, or atryptophan substitution to a stop codon at amino acid position 517(W517*) of SEQ ID No. 3, or a guanine substitution to an adenine atnucleotide position 1538 of SEQ ID No.
 15. 13. The wheat plant of claim12, wherein the at least one mutation in the B genome of the Lpx-B1.2gene comprises a tryptophan substitution to a stop codon at amino acidposition 510 (W510*) of SEQ ID No.
 6. 14. The wheat plant of claim 12,wherein the at least one mutation in the D genome of the Lpx-D1 genecomprises a tryptophan substitution to a stop codon at amino acidposition 494 (W494*) of SEQ ID No.
 3. 15. Wheat grain from the wheatplant of claim
 12. 16. Flour comprising wheat grain of claim
 15. 17. Awheat seed, plant part or progeny thereof from a wheat plant of claim12, wherein said progeny comprises the Lpx1 mutations.
 18. The wheatplant of claim 12, wherein the at least one mutation in the B genome ofthe Lpx-B1.2 gene comprises a tryptophan substitution to a stop codon atamino acid position 510 (W510*) of SEQ ID No. 6 and the at least onemutation in the D genome of the Lpx-D1 gene comprises a tryptophansubstitution to a stop codon at amino acid position 494 (W494*) of SEQID No.
 3. 19. The wheat plant of claim 18, wherein milled grain fromsaid wheat plant has a property selected from the group consisting of:(a) increased shelf-life; (b) increased oxidative stability; (c)decreased production of Lpx1 protein; (d) decreased activity of the Lpx1protein; (e) decreased hexanal production; (f) decreased pinellic acidproduction; (g) decreased decomposition products from fatty acids; or(h) improved sensory characteristics as compared to milled grain from awild type wheat plant.
 20. Wheat grain from the wheat plant of claim 18.21. Flour comprising wheat grain of claim
 20. 22. A wheat seed, plantpart or progeny thereof from a wheat plant of claim 18, wherein saidprogeny comprises the Lpx1 mutations.
 23. The wheat plant of claim 12,wherein milled grain from said wheat plant has a property selected fromthe group consisting of: (a) increased shelf-life; (b) increasedoxidative stability; (c) decreased production of Lpx1 protein; (d)decreased activity of the Lpx1 protein; (e) decreased hexanalproduction; (f) decreased pinellic acid production; (g) decreaseddecomposition products from fatty acids; or (h) improved sensorycharacteristics as compared to milled grain from a wild type wheatplant.